Phenolic Compositions Derived From Apple Skin And Uses Thereof

ABSTRACT

Described herein are phenolic compositions derived from apple skins. In particular, described herein are flavonoid-rich fractions derived from apple skin extract. The compositions are useful in the prevention and treatment of conditions associated with oxidative stress and/or inflammation, including certain neurodegenerative diseases. Methods of producing the compositions are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 61/333,091 filed May 10, 2010, which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The disclosure relates to phenolic compositions derived from apple skinand uses thereof in the prevention and treatment diseases and conditionsassociated with oxidative stress and/or inflammation.

BACKGROUND

Natural plant-based phenolic compounds, such as flavonoids, havereceived much attention for their potent antioxidant properties.Phenolic compounds are present in many plant sources, such as fruits,vegetables and many herbal or aromatic plants. Given their potentanti-oxidant activities, plant phenolics have been under investigationas health-promoting substances for over 20 years.

Apples are a good dietary source of phenolic compounds. Apple peel has3- to 6-fold higher flavonoid content than apple flesh and has uniqueflavonoids, such as quercetin glycosides, not found in the flesh (Wolfe,Wu & Liu, 2003; Wolfe & Liu, 2003).

Apple peel extracts have been shown to possess powerful free radicalscavenging activity (Kondo et al., 2002). Various apple peel extractsand methods of making them are described, for example, in US2005/0147723 A1, US 2006/0172012 A1, U.S. Pat. No. 6,440,410 B1, EP 0659 347 A1, and Kim et al., 2005. PCT application WO 2009/0767776 A1 toRupasinghe et al. discloses a crude extract derived from apple peel andits use in preventing oxidation of polyunsaturated fatty acids or lipidsin food products.

Oxidative stress induced by reactive oxygen species is linked to anumber of human conditions and diseases. Plant-derived antioxidants maytherefore provide dietary modulators useful in preventing or treating anumber of such diseases, including certain neurodegenerative diseases(Kaur and Kapoor, 2001; Heo et al. 2004). A ubiquitous flavonoid,quercetin 3-O-glucoside (Q3G), was recently suggested to haveneuroprotective effects in vitro (Soundararajan et al., 2008). Directtreatment of neuronal cells in vivo is not a likely therapeutic approachhowever, and there is no indication that apple-derived extracts wouldhave any beneficial effect in vivo.

The costs associated with neurodegenerative disorders are in excess of$20 billion annually in North America. Currently, there are noregistered natural products with health claims for protecting againstthese neurodegenerative disorders. As our overall population ages, thereis increased desire to maintain health as well as to use alternativemedications derived from natural or biological sources. There is a needfor discovery and development of safe and effective natural products totreat, prevent or reduce the risk of oxidative stress-mediatedconditions and diseases, including neurodegenerative disorders.

Apple peels are a waste product of the apple processing industry in manycountries and therefore represent and attractive resource forvalue-added health-promoting products. While crude extracts have beenprepared, there is a need for more refined compositions having potentantioxidant properties. Extraction of phenolic compounds from plantsources presents challenges, since the phenolic compounds are easilyoxidized and degrade under harsh extraction conditions.

SUMMARY OF THE ASPECTS

Compositions rich in biologically active phenolic compounds were derivedfrom the skins of apples and were analyzed for phenolic content andprofile. It was demonstrated that the phenolic compositions describedherein are protective against neuronal cell death in vitro and braindamage and disability in vivo associated with oxidative stress. Thephenolic compositions described herein are expected to be useful in thetreatment and/or prevention of diseases or conditions associated withoxidative stress, including certain neurodegenerative diseases. Variousnon-limiting aspects and embodiments are described below.

In one aspect, there is provided a composition for use in preventing ortreating an oxidative-stress mediated disease or condition, thecomposition comprising a phenolic extract or fraction thereof derivedfrom apple skin. In some embodiments, the phenolic extract or fractionthereof comprises a flavonol component, an anthocyanin component, adihydrochalcone component, a phenolic acid component, and a flavan-3-olcomponent.

In some embodiments, the flavonol component comprises quercetin,Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside,Q-3-O-rhamnoside or a combination thereof. In some embodiments, theanthocyanin component comprises cyanidin-3-O-galactoside. In someembodiments, the dihydrochalcone component comprises phloridzin,phloritin or a combination thereof. In some embodiments, the phenolicacid component comprises chlorogenic acid, cafeic acid, ferulic acid,isoferulic acid or a combination thereof. In some embodiments, theflavan-3-ol component comprises epigallocatechin, catechin, epicatechinor a combination thereof.

In one embodiment, the phenolic extract or fraction thereof comprises: aflavonol component comprising quercetin, Q-3-O-paltoside,Q-3-O-rutinoside, Q-3-O galactoside, Q-3-O-glucoside andQ-3-O-rhamnoside; an anthocyanin component comprisingcyanidin-3-β-galactoside; a dihydrochalcone component comprisingphloridzin and phloritin; a phenolic acid component chlorogenic acid,cafeic acid, ferulic acid and isoferulic acid; and a flavan-3-olcomponent comprising epigallocatechin, catechin and epicatechin.

In some embodiments, the extract or fraction thereof comprises: fromabout 10.0% to about 60.0%, from about 15.0% to about 50.0%, from about20.0% to about 50.0%, from about 20.0% to about 35.0%, from about 20.0%to about 30.0%, from about 20.0% to about 25.0% Q-3-O-galactoside; fromabout 10.0% to about 60.0%, from about 15.0% to about 50.0%, from about20.0% to about 50.0%, from about 20.0% to about 40.0%, from about 20.0%to about 35.0%, from about 20.0% to about 30.0%, from about 20.0% toabout 25.0%, from about 30.0% to about 35.0% Q-3-O-rhamnoside; fromabout 1.0% to about 20.0%, from about 5.0% to about 15.0%, from about7.0% to about 13.0%, from about 5.0% to about 10.0%, from about 10.0% toabout 15.0%, Q-3-O-rutinoside; from about 1.0% to about 20.0%, fromabout 5.0% to about 15.0%, from about 7.0% to about 13.0%, from about10.0% to about 15.0%, from about 10.0% to about 15.0%, Q-3-O-glucoside;from about 0.5% to about 10.0%, from about 0.5% to about 5.0%, fromabout 0.5% to about 2.5, from about 0.5% to about 2.0%, from about 1.0%to about 2.0%, from about 2.0% to about 6.0%, from about 3.5% to about5.5%, from about 4.0% to about 5.0% cyanidin-3-O-galactoside; from about0.5% to about 10.0%, from about 1.0% to about 10.0%, from about 2.0% toabout 10.0%, from about 1.0% to about 5.0%, from about 1.5% to about4.5%, from about 3.5% to about 7.5%, about 3.0% to about 6.0%, about 3%,about 5% phloridzin; from about 1.0% to about 20.0%, from about 2.0% toabout 15.0%, about 5.0% to about 15.0%, from about 2.5% to about 6.5%,from about 8% to about 12.0%, about 4% or about 10% chlorogenic acid;and from about 1.0% to about 20.0%, from about 1.0% to about 15.0%, fromabout 5.0% to about 15.0%, from about 2.5% to about 10.0%, from about2.5% to about 6.5%, from about 5.0% to about 10.0%, from about 8.0% toabout 10.0% epicatechin.

In some embodiments, the extract or fraction thereof comprises: fromabout 20.0% to about 30.0% Q-3-O-galactoside; from about 20.0% to about30.0% Q-3-O-rhamnoside; from about 10.0% to about 15.0%,Q-3-O-rutinoside; from about 10.0% to about 15.0%, Q-3-O-glucoside; fromabout 2.0% to about 6.0% cyanidin-3-O-galactoside; from about 1.0% toabout 5.0%, phloridzin; from about 8% to about 12.0% chlorogenic acid;and from about 5.0% to about 10.0% epicatechin.

In some embodiments, the extract or fraction thereof comprises: fromabout 20.0% to about 35.0% Q-3-O-galactoside; from about 20.0% to about35.0% Q-3-O-rhamnoside; from about 5.0% to about 15.0% Q-3-O-rutinoside;from about 5.0% to about 15.0% Q-3-O-glucoside; from about 0.5% to about2.5 cyanidin-3-O-galactoside; from about 1.0% to about 5.0% phloridzin;from about 5.0% to about 15.0% chlorogenic acid; and from about 5.0% toabout 10.0% epicatechin.

In some embodiments, the phenolic extract is obtainable by an aqueousextraction process having the following steps: obtaining a sample ofapple skins; treating the skins to inhibit degradation of phenoliccompounds; optionally dehydrating the skins and converting the skins toa powder form; extracting the skins one or more times with a food-gradesolvent, such as, ethanol; optionally subjecting the skins to sonicationduring extraction; removing solids to obtain a phenolic extract;optionally concentrating the phenolic extract; optionally removingsugars from the phenolic extract, and optionally concentrating, dryingand/or freezing the phenolic extract.

In some embodiments, the fraction is obtainable by fractionation of thephenolic extract using a suitable eluent, followed by selection of afraction having a high phenolic content, In some embodiments, a fractionhaving a high flavonol content is selected.

In some embodiments, the fraction is eluted in about 40% to about 60%ethanol. In some embodiments, the fraction is eluted in about 45% toabout 50% ethanol.

In some embodiments, the fraction has the following phenolic profile:from about 60.0% to about 95.0% flavonol selected from the groupconsisting of quercetin (O), Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-0galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside and combinations thereof;from about 0.5% to about 10.0% cyanidin-3-O-galactoside; from about 1.0%to about 10.0 dihydrochalcone selected from the group consisting ofphloridzin, phloritin and combinations thereof; from about 1.0% to about20.0% phenolic acid selected from the group consisting of chlorogenicacid, cafeic acid, ferulic acid, isoferulic acid and combinationsthereof; and from about 1.0% to about 20.0% flavan-3-ol selected fromthe group consisting of epigallocatechin, catechin, epicatechin andcombinations thereof, wherein the percentages are based the total weightof phenolic content of the fraction and wherein the total does notexceed 100%.

In some embodiments, the fraction has the following phenolic profile:from about 70.0% to about 90.0% flavonol selected from the groupconsisting of quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside and combinationsthereof; from about 0.5% to about 5.0% cyanidin-3-O-galactoside; fromabout 2.0% to about 10.0% dihydrochalcone selected from the groupconsisting of phloridzin, phloritin and combinations thereof; from about2.0% to about 15.0% phenolic acid selected from the group consisting ofchlorogenic acid, cafeic acid, ferulic acid, isoferulic acid andcombinations thereof; and from about 1.0% to about 15.0% flavan-3-olselected from the group consisting of epigallocatechin, catechin,epicatechin and combinations thereof.

In some embodiments, the fraction comprises: from about 80.0% to about90.0% flavonol selected from the group consisting of quercetin,Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside,Q-3-O-rhamnoside and combinations thereof; from about 0.5% to about 2.5%cyanidin-3-O-galactoside; from about 3.5% to about 7.5% dihydrochalconeselected from the group consisting of phloridzin, phloritin andcombinations thereof from about 2.5% to about 6.5% phenolic acidselected from the group consisting of chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid and combinations thereof and from about2.5% to about 6.5% flavan-3-ol selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof.

In some embodiments, the fraction comprises: from about 70.0% to about80.0% flavonol selected from the group consisting of quercetin,Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside,Q-3-O-rhamnoside and combinations thereof from about 2.0% to about 6.0%cyanidin-3-O-galactoside; from about 1.5% to about 4.5% dihydrochalconeselected from the group consisting of phloridzin, phloritin andcombinations thereof from about 8.0% to about 12.0% phenolic acidselected from the group consisting of chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid and combinations thereof and from about8.0% to about 12.0% flavan-3-ol selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof.

In some embodiments, the composition further comprises apharmaceutically acceptable excipient.

In some embodiments, the disease or condition associated with oxidativestress or inflammation is aging, obesity, autoimmune diseases, such as,arthritis, diabetes, lupus, colitis and Crohn's disease, heart disease,atherosclerosis, stroke, myocardial infarction, retinal degeneration,hearing loss, fragile X syndrome, chronic fatigue syndrome, traumaticbrain injury, spinal cord injury, head injury, demyelinating disorders,such as multiple sclerosis, devic's, progressive multifocalleukoencephalopathy, optic neuritis, leukodystrophies, charcot-marietooth, and guillian-barre syndrome, or oxidative stress-inducedneurodegenerative diseases, such as multiple sclerosis, Parkinson'sdisease and Alzheimer's disease and vascular dementia. The compositionsdescribed herein may also be useful in the treatment or prevention ofcertain oxidative stress-mediated orphan disorders, such as amyotrophiclateral sclerosis (ALS), primary progressive MS, charcot-marie-toothdisease, and spinal muscular atrophy.

In another aspect, there is provided a method of preventing or treatingan oxidative-stress mediated disease or condition, comprisingadministering to a subject an effective amount of a composition asdefined herein.

In another aspect, there is provided a method of preventing or treatingan oxidative-stress mediated disease or condition, comprisingadministering to a subject an effective amount of a phenolic extract orfraction thereof derived from apple skin.

In some embodiments, the phenolic extract or fraction thereof isadministered to a subject in multiple doses.

In some embodiments, the phenolic extract or fraction thereof isadministered orally.

In some embodiments, the phenolic extract or fraction thereof isadministered in the form of a concentrate, a liquid, a powder, anemulsion, a suspension, a paste, a gel, a film, a gum, a drop, a tablet,a capsule, a microcapsule, a food additive.

In another aspect, there is provided a fraction of a phenolic apple skinextract, the extract obtainable by an aqueous extraction process, thefraction obtainable by fractionating the extract in a chromatographycolumn using a suitable eluent and selecting a fraction having a highphenolic content, wherein the fraction comprises a flavonol component,an anthocyanin component, a dihydrochalcone component, a phenolic acidcomponent, and a flavan-3-ol component. The fraction has the features asdefined above.

In another aspect, there is provided a dietary supplement or naturalhealth product for preventing or reducing damage due to oxidative stresscomprising a fraction of as defined herein.

In another aspect, there is provided a functional food or beveragecomprising a fraction as defined herein.

In another aspect, there is provided a pharmaceutical compositioncomprising a fraction as defined herein together with a pharmaceuticallyacceptable excipient.

In some embodiments, the pharmaceutical composition is formulated forenteral administration, topical administration, parenteraladministration, intrapulmonary administration or nasal administration.In some embodiments, the enteral administration is oral administration.

In some embodiments, the pharmaceutical composition is for use in theprevention or treatment of a disease or condition associated withoxidative stress.

In some embodiments, the pharmaceutical composition is for use in theprevention or treatment of a disease or condition associated withinflammation.

In another aspect there is provided, a use of a composition as definedherein the preparation of a medicament for the treatment or preventionof a disease or condition associated with oxidative stress and/orinflammation.

In another aspect there is provided, use of a composition as definedherein for the treatment or prevention of a disease or conditionassociated with oxidative stress and/or inflammation.

In another aspect there is provided, a commercial package comprising acomposition as defined herein together with instructions for use as adietary supplement or natural health product.

In another aspect there is provided a commercial package comprising thedietary supplement or natural health product as defined herein togetherwith instructions for use in promoting health.

In another aspect there is provided a commercial package comprising apharmaceutical composition as defined herein together with instructionsfor use in the use in the treatment or prevention of a disease orcondition associated with oxidative stress and/or inflammation.

In another aspect there is provided a food additive comprising afraction as defined herein.

In another aspect there is provided a cosmetic product comprising afraction as defined herein.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1 illustrates that hypoxic-ischemic (HI) brain injury in micetreated with F5 had better motor performance as assessed by the rotarodcompared to control mice treated with vehicle.

FIG. 2 illustrates that mice treated with F5 were protected from thehemisphere loss caused HI brain injury compared to vehicle treated mice.

FIG. 3 illustrates that F5 treatment increased the number of viableneurons in the striatum after HI brain injury compared to vehicletreatment.

FIG. 4 illustrates that a single F5 treatment did not significantlyprevent cell loss in the hippocampus after HI brain injury in comparisonto the vehicle control group.

FIG. 5 is a photomicrograph showing brain volume in vehicle (water)- andF5-treated mice and after HI brain injury;

FIG. 6 is a photomicrograph showing striatal damage in vehicle (water)-and F5-treated mice after HI brain injury.

FIG. 7 illustrates that F4 reduced the death of primary cortical neuronssubjected to oxygen glucose deprivation in a comparison of F4, F4components and F4 metabolites.

FIG. 8 illustrates a dose-dependent reduction in HI-induced hippocampalneuron loss produced by oral (p.o.) administration of F4.

FIG. 9 illustrates a dose-dependent reduction of HI-induced hippocampaldamage by a purified apple fraction F4.

FIG. 10 illustrates a dose-dependent reduction of HI-induced striatalneuron loss produced by F4.

FIG. 11 illustrates a dose-dependent reduction of HI-induced striatalneuron loss produced by F4.

FIG. 12 illustrates a failure of one dose of F4 (25 mg/kg, p.o.) beforeHI to reduce striatal neuron loss.

FIG. 13 illustrates a reduction of striatal neuron loss by three dosesof F4 (25 mg/kg/day, p.o) before HI.

FIG. 14 illustrates a reduction of striatal neuron loss by 7 doses of F4(25 mg/kg/day for 7 days, p.o.) before HI.

FIG. 15 illustrates that one dose of F4 (25 mg/kg, p.o.) before HI didnot reduce hippocampal damage.

FIG. 16 illustrates that three doses of F4 (25 mg/kg, p.o.) before HIreduced hippocampal tissue loss.

FIG. 17 illustrates that seven doses of F4 (25 mg/kg, p.o.) before HIreduced hippocampal tissue loss.

FIG. 18 illustrates that oral administration of F4 (25 mg/kg p.o)beginning 24 hours after first clinical signs of EAE reduced diseaseprogression compared to animals that received water.

FIG. 19 illustrates that oral administration of F4 (50 m/kg) reducesproduction of the inflammatory cytokine tumour necrosis factor alpha(TNFα) in LPS-stimulated whole blood from mice subjected to HI. A,LPS-induced TNF-α release was the same in whole blood from sham treatedanimals (no brain injury) received oral administration of water or F4.B, LPS-induced TNF-α release was the greater in whole blood from HIanimals (brain injury) that received oral administration of watercompared to mice that received F4.

DETAILED DESCRIPTION

Apples, particularly apple skins, are a rich source of phenoliccompounds, such as flavonoids, which have potent antioxidant potential.Apple skins are a waste product of the apple processing industry (e.g.apple sauce, apple pie) in many countries and are typically available athigh quantity and low cost. Therefore, phenolic compounds isolated fromapple skin represent an ideal source of natural antioxidants for thefood, natural health product, pharmaceutical and cosmetic industries.Since the raw source material is readily available at low cost, aneconomic benefit is realized which can be passed on to consumers,thereby having the potential to decrease the cost of health care.

The present disclosure relates to phenolic compositions, in particular,phenolic extracts and fractions thereof derived from apple skin, andtheir use in the prevention or treatment of diseases or conditionsassociated with oxidative stress and/or inflammation. Also disclosedherein are methods of preparing the phenolic compositions from appleskins. The phenolic compositions are naturally-derived,consumer-friendly compositions, thereby fulfilling a growing desire fornatural health-promoting products, such as natural health products ornutriceuticals, dietary supplements, and functional foods. Thecompositions have potent antioxidant and anti-inflammatory propertiesmaking them also suitable for use in pharmaceutical and cosmeticapplications.

Based on the results discussed further below, it is expected that thephenolic compositions described herein will have utility in theprevention or treatment of various conditions and diseases associatedwith oxidative stress or inflammation. With regards to oxidative stress,this hypothesis is supported by the fact that free radical generationhas been implicated in numerous human disorders and conditions, andflavonoids are excellent free radical scavengers. Oxidative stressgenerally refers to excess production of free radicals, which can resultfrom a number of different causes, such as tissue damage, inflammation,and excitotoxicity. The beneficial effects of two exemplarycompositions, F4 or F5, were seen at concentrations less than 1/10 thatof an anti-oxidant concentration of Vitamin C, thus the beneficialeffects of the compositions disclosed herein are not likely entirely dueto anti-oxidant effects. There is an emerging view that flavonoids, andtheir in vivo metabolites, may exert modulatory actions in cells throughactions at protein kinase and lipid kinase signalling pathways.Flavonoids, and more recently their metabolites, have been reported toact at phosphoinositide 3-kinase (PI 3-kinase), Akt/protein kinase B(Akt/PKB), tyrosine kinases, protein kinase C (PKC), and mitogenactivated protein kinase (MAP kinase) signalling cascades. Inhibitory orstimulatory actions at these pathways are likely to affect cellularfunction profoundly by altering the phosphorylation state of targetmolecules and by modulating gene expression (Williams R J et al, 2004).Thus, in addition to being radical scavengers, flavonoids may alsomodulate various kinase pathways involved in cell stress and cell death.With regard to inflammation, the results demonstrated herein illustratethat the phenolic compositions of the present disclosure are alsocapable of inhibiting phosphodiesterases and inflammatory cytokineproduction, indicating that the compositions are effectiveanti-inflammatory agents as well. The compositions defined herein haveunique phenolic profiles and it is believed that synergies between theparticular components present in a given fraction may contribute totheir potent therapeutic effects.

The phenolic compositions disclosed herein have potential for preventingor treating one or more conditions or diseases associated with oxidativestress or inflammation, including those having components related toapoptosis, necrosis, damage to cerebral vasculature, neuroinflammation,excitotoxicity, and the like. Exemplary diseases and conditionsassociated with oxidative stress and/or inflammation include, but arenot limited to, aging, chronic fatigue syndrome, neurodegenerativedisorders, autoimmune disorders, metabolic disorders, and vasculardisorders. Neurodegenerative disorders include, for example, Parkinson'sdisease, Alzheimer's disease, retinal degeneration, hearing loss,fragile X syndrome, chronic fatigue syndrome, traumatic brain injury,spinal cord injury, head injury and demyelinating disorders.Demyelinating disorders include, for example, multiple sclerosis,devic's, progressive multifocal leukoencephalopathy, optic neuritis,leukodystrophies, charcot-marie tooth, and guillian-barre syndrome.Autoimmune disorders include, for example, type I diabetes, rheumatoidarthritis, lupus, colitis and Crohn's disease. Vascular disordersinclude, for example, stroke, atherosclerosis, myocardial infarction andvascular dementia. Beneficial effects of flavonoids may derive, at leastin part, from protection of the cerebral vasculature andmicrovasculature, such as in stroke, MS, and vascular dementia.Metabolic disorders include, for example, obesity, type II diabetes, andlipid disorders, such as, hypercholesteremia. The compositions describedherein may also be useful in the treatment or prevention of certainoxidative stress-mediated orphan disorders, such as amyotrophic lateralsclerosis (ALS), primary progressive MS, charcot-marie-tooth disease,and spinal muscular atrophy. It will be understood that diseases andconditions associated with oxidative stress and/or inflammation may fallinto more than one category listed above.

The following section defines various terms and expressions usedthroughout the instant specification.

The term “phenolic” refers generally to naturally-occurring chemicalcompounds found in plants having at least one phenol group. As usedherein, “phenolic” refers to polyphenolic compounds derived from apples,in particular, flavonoids present in apple skins (see, for example,Table 2a and b). Flavonoids include, but are not limited to, flavonols(e.g. quercetin and various glycosides thereof), anthocyanidins,dihydrochalcones, phenolic acids, and flavan-3-ols (or catechins).Flavonoids are known to scavenge free radicals, inhibit a variety ofkinases, reduce lipid peroxidation, inhibit apoptosis, prevent plateletaggregation and exhibit anti-inflammatory effects.

As used herein, “phenolic content” of an extract or fraction thereofrefers to monomeric phenolic content and is based on the monomericphenolic compounds recited in Table 2a and 2b, where the percentage ofeach compound is calculated based the total weight of the monomericphenolic content of the composition. The total percentage of thephenolic content does not exceed 100%. The compositions may compriseadditional phenolic compounds not recited in Tables 2a and 2b but theseare not included for the purpose of determining the percentages usedherein.

A “high phenolic content” refers to selection of a fraction having arelatively high phenolic content compared to other fractions isolated,or compared to a crude extract. For example, of 15 fractions analyzed inTables 2a and 2b, fractions F4 and F5 have relatively high monomericphenolic content compared to the other fractions. In particular, F4 andF5 have a relatively high flavonol content compared to the otherfractions. A “high flavonol content” refers to a fraction that isenriched in flavonols compared to other fractions or compared to a crudeextract. Through a process such as fractionation, a fraction may have ahigher flavonol content (% flavonol in the total phenolic content) thana crude extract from which is was derived.

A “phenolic extract or fraction thereof” is a flavonoid rich extractprepared from extraction of apple skills, or an enriched fractionisolated therefrom. A desired fraction may be isolated, for example,using column chromatography. The extract or fraction thereof may befurther processed to any desired form and, for example, may be in theform of a liquid, a suspension, an emulsion, a solution, or a solid(such as a powder or a lyophilized product). Thus, it will be understoodthat the term extract or fraction does not necessarily refer to a liquidform of the extract or fraction.

As used herein, the “phenolic profile” of an extract or a fractionrefers to the particular combination of phenolic compounds, inparticular, flavonoids, in the extract or fraction and their amountrelative to each other.

The term “effective amount” is an amount sufficient to achieve a desiredoutcome in a subject. For treatment, an effective amount is atherapeutically effective amount. The therapeutically effective amountcan vary depending, for example, on the disease, disorder, or symptom ofthe disease or disorder, severity of the disease, disorder, or symptomof the disease or disorder, the age, weight, or health of the patient tobe treated, and the judgment of the prescribing physician. Anappropriate therapeutically effective amount in any given instance maybe ascertained by those skilled in the art or capable of determinationby routine experimentation. In some cases, a therapeutically effectiveamount is an amount sufficient to provide at least about 1 mg/kg/d toabout 2000 mg/kg/d, 1 mg/kg/d to about 1500 mg/kg/d or 1 mg/kg/d toabout 1000 mg/kg/d, or about 5 mg/kg/d to about 500 mg/kg/d, or about 25mg/kg/d to about 200 mg/kg/d, or about 10 mg/kg/d to about 100 mg/kg/d,of total phenolic content to the subject. For prevention, an effectiveamount is a prophylactically effective amount. An effective amount mayalso be an amount sufficient to promote good heath. The effective amountwill depend, at least in part, on the particular use of the phenoliccomposition (e.g. dietary supplement, functional food, natural healthproduct or pharmaceutical product) and the desired outcome.

The term “oxidative stress” refers generally to an imbalance between theproduction of reactive oxygen species and a biological system's abilityto readily detoxify the reactive intermediates or easily repair theresulting damage. In humans, oxidative stress is implicated in manyconditions and diseases, such as aging, arthritis, diabetes, heartdisease, atherosclerosis, stroke, myocardial infarction, vasculardementia, retinal degeneration, hearing loss, fragile X syndrome,chronic fatigue syndrome and neurodegenerative disease.

As used herein, the term “disease or condition” refers to a disease,disorder, condition, pathology, or symptom of any of the foregoing. Theterm “disease” typically refers to an abnormal condition affecting thebody of an organism. It is often construed to be a medical conditionassociated with specific symptoms and signs. As used herein, a“condition” refers to a state of being which is desired to be treated orprevented but which is not typically regarded as a disease, such asaging.

As used herein, the term “treat” or “treating” means to alleviate oreliminate symptoms, either on a temporary or permanent basis, or to slowthe appearance of symptoms of a disease or condition, or to slow thedevelopment of a disease or condition, or to prolong the period beforerecurrence of a symptom or negative event associated with a disease orcondition. The act of treating may not eliminate symptoms altogether butwill provide some relief or improvement to the subject as compared to notreatment.

As used herein, the term “prevent” or “preventing” means to prevent theonset of a disease or condition, or a symptom of a disease or condition,for example, in a population believed to be susceptible to the diseaseor condition.

As used herein, the term “excipients” refers to carriers, diluents,additives and the like, having substantially no pharmacologicalactivity. The excipients are preferably “pharmaceutically acceptable”referring to excipients which are nontoxic when administered to asubject in an amount sufficient to provide a desired effect and which donot destroy the biological activity of the phenolic extract or fractionthereof.

As sued herein, a “subject” refers to mammals, in particular, humans,domesticated animals (e.g. pets), laboratory animals, and livestock.

A “dose” refers to the amount of active agent to be administered to asubject in a given unit(s) of a dosage form. The dose required toachieve efficacy can vary depending on, for example, the disease orcondition to be treated, the dosage form, and the route ofadministration.

As used herein, “food” encompasses any item consumable by humans oranimals, for example, for nutrition, health or pleasure, and includesboth foods and beverages.

Terms of degree such as “substantially”, “about” and “approximately” asused herein mean a reasonable amount of deviation of the modified termsuch that the end result is not significantly changed.

As used herein, the term “about” in association with a numeric value orrange refers to a variation of +/−5%.

When introducing elements disclosed herein, the articles “a”, “an”,“the”, and “said” are intended to mean that there are one or more of theelements.

The terms “comprising”, “having”, “including” are intended to beopen-ended and mean that there may be additional elements other than thelisted elements.

Reference is now made in detail to embodiments of the presentdisclosure. The disclosed embodiments are not intended to be limiting ofthe claims. To the contrary, the claims are intended to coveralternatives, modifications, and equivalents.

In one broad aspect, there is provided herein a composition for use inpreventing or treating a disease or condition associated with oxidativestress and/or inflammation. The composition comprises a phenolic extractor fraction thereof derived from apple skin.

The flavonoid-rich phenolic extract or fraction thereof comprises aflavonol component, an anthocyanin component, a dihydrochalconecomponent, a phenolic acid component, and a flavan-3-ol component. Thecomponents are used in measuring total phenolic content of the extractor fraction thereof.

The flavonol component may comprise quercetin, Q-3-O-paltoside,Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnosideor a combination thereof. In one embodiment, wherein the flavonolcomponent comprises quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside and Q-3-O-rhamnoside.

The anthocyanin component may comprise cyanidin-3-O-galactoside.

The dihydrochalcone component may comprise phloridzin, phloritin or acombination thereof. In one embodiment, the dihydrochalcone componentcomprises phloridzin and phloritin.

The phenolic acid component may comprise chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid or a combination thereof. In oneembodiment, wherein the phenolic acid component comprises chlorogenicacid, cafeic acid, ferulic acid and isoferulic acid.

The flavan-3-ol component may comprises epigallocatechin, catechin,epicatechin or a combination thereof. In one embodiment, the flavan-3-olcomponent comprises epigallocatechin, catechin and epicatechin.

In one embodiment, the phenolic extract or fraction thereof comprises: aflavonol component comprising quercetin, Q-3-O-paltoside,Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside andQ-3-O-rhamnoside; an anthocyanin component comprisingcyanidin-3-O-galactoside; a dihydrochalcone component comprisingphloridzin and phloritin; a phenolic acid component chlorogenic acid,cafeic acid, ferulic acid and isoferulic acid; and a flavan-3-olcomponent comprising epigallocatechin, catechin and epicatechin.

The phenolic extract is obtainable by an aqueous extraction process. Anexemplary process has the following steps: obtaining a sample of appleskins; treating the skins to inhibit degradation of phenolic compounds;optionally dehydrating the skins and converting the skins to a powderform; extracting the skins one or more times with a food-grade solvent,such as, ethanol; optionally subjecting the skins to sonication duringextraction; removing solids to obtain a phenolic extract; optionallyconcentrating the phenolic extract; optionally removing sugars from thephenolic extract, and optionally concentrating, drying and/or freezingthe phenolic extract.

In some embodiments, the extract is further purified, for instance usingcolumn chromatography (e.g. C18 column chromatography). Purification maybe performed to remove additional components from the crude extract, forexample, highly lipophilic components. Ethanol or another suitablefood-grade solvent can be used as the eluent, for example, 80%, 85%,90%, 95% or 100% ethanol.

In some embodiments, the extract (crude or purified) is subjected tofractionation to obtain eluted fractions. Fractionization may be carriedout using any suitable method, such as column chromatography. Preferablythe selected fraction has a relatively high phenolic content compared toother fractions potentially isolated. An eluted fraction may comprise aunique combination and/or concentration of individual componentscompared to the crude extract and compared to other fractions (i.e. aunique phenolic profile). Thus, different fractions may exhibitdifferent biological effects. In addition, each fraction prepared maycontain unidentified active compounds that may contribute to thebiological activity. Synergies may be seen between the activecomponents. In some embodiments, fractions enriched for flavonols areselected.

In some embodiments, the fraction is obtainable by fractionation of thephenolic extract in a chromatography column using a suitable eluent. Insome embodiment, the fraction is obtained by fractionation of thephenolic extract in a chromatography column using a suitable eluent. Anysuitable eluent may be used, for example, ethanol or another suitablefood-grade eluent. In some embodiments, the chromatography used is flashchromatography with a C18 column using a polymeric sorbent.

In some embodiments, the fractionation step is followed by analysis ofthe fractions and selection of one or more fractions having a highphenolic content. Phenolic content may be measured by any suitablemeans. Is some embodiments, the phenolic content is measured asdescribed in Example 1. In some embodiments, the selected fraction has ahigh phenolic content of greater than about 7000 mg/ml, greater thanabout 9000 mg/ml, greater than about 10000 mg/mL, greater than about11000 mg/mL, greater than about 12000 mg/mL, or greater than about 13000mg/mL based on an elution volume of 800 ml.

Is some embodiments, the selected fraction has a high flavonol contentfor example, a flavonol content of greater than about 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% based the total phenolic content of thefraction. The flavonol content may be relatively high compared to otherfractions and/or compared to a crude extract.

In some embodiments, the selected fraction is a fraction or fractionsthat elute in about 40% to about 60% ethanol, as described in Example 1.For example, one or more fractions eluting in ethanol concentrations maybe removed prior to collecting one or more fractions that elute betweenabout 40% to about 60% ethanol. In some embodiments, the fraction iseluted in about 45% to about 50% ethanol. In one embodiment, thefraction is eluted in about 45% ethanol. In another embodiment, thefraction is eluted in about 50% ethanol.

In some embodiments, the composition comprises fraction F4 or F5 ofTable 2a, which may optionally be diluted, concentrated or dried priorto use. In some embodiments, F4 and F5 are combined to form a singleflavonol-rich fraction that elutes between about 45% to about 50%ethanol.

In some embodiments, the extract or fraction thereof comprises fromabout 60.0% to about 95.0%, from about 70.0% to about 90.0%, from about70.0% to about 80.0%, from about 80.0% to about 90.0% flavonol. In someembodiments, the extract or fraction thereof comprises from greater thanabout 60.0%, 70%, 75%, 80%, 85%, 90% or 95.0% flavonol. This is higherthan the crude extract, which was found to have a flavonol concentrationof only about 50%. In some embodiments, the flavonol is selected fromthe group consisting of quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside and combinationsthereof.

In some embodiments, the extract or fraction thereof comprises fromabout 10.0% to about 60.0%, from about 15.0% to about 50.0%, from about20.0% to about 50.0%, from about 20.0% to about 35.0%, from about 20.0%to about 30.0%, from about 20.0% to about 25.0% Q-3-O-galactoside.

In some embodiments, the extract or fraction thereof comprises fromabout 10.0% to about 60.0%, from about 15.0% to about 50.0%, from about20.0% to about 50.0%, from about 20.0% to about 40.0%, from about 20.0%to about 35.0%, from about 20.0% to about 30.0%, from about 20.0% toabout 25.0%, from about 30.0% to about 35.0% Q-3-O-rhamnoside.

In some embodiments, the extract or fraction thereof comprises fromabout 1.0% to about 20.0%, from about 5.0% to about 15.0%, from about7.0% to about 13.0%, from about 5.0% to about 10.0%, from about 10.0% toabout 15.0%, Q-3-O-rutinoside.

In some embodiments, the extract or fraction thereof comprises fromabout 1.0% to about 20.0%, from about 5.0% to about 15.0%, from about7.0% to about 13.0%, from about 10.0% to about 15.0%, from about 10.0%to about 15.0%, Q-3-O-glucoside.

In some embodiments, the extract or fraction thereof comprises fromabout 0.5% to about 10.0%, from about 0.5% to about 5.0%, from about0.5% to about 2.5, from about 0.5% to about 2.0%, from about 1.0% toabout 2.0%, from about 2.0% to about 6.0%, from about 3.5% to about5.5%, from about 4.0% to about 5.0% cyanidin-3-β-galactoside.

In some embodiments, the extract or fraction thereof comprises fromabout 0.5% to about 10.0%, from about 1.0% to about 10.0%, from about2.0% to about 10.0%, from about 1.5% to about 4.5%, from about 3.5% toabout 7.5%, about 3.0% to about 6.0%, about 3%, about 5%dihydrochalcone. In some embodiments, the dihydrochalcone is selectedfrom the group consisting of phloridzin, phloritin and combinationsthereof. In some embodiments, phloridzin represents a major proportionof dihydrochalcone in the composition, for example, greater than 99%phloridzin (300:1, 500:1, 700:1).

In some embodiments, the extract or fraction thereof comprises fromabout 0.5% to about 10.0%, from about 1.0% to about 10.0%, from about2.0% to about 10.0%, from about 1.5% to about 4.5%, from about 3.5% toabout 7.5%, about 3.0% to about 6.0%, about 3%, about 5% phloridzin.

In some embodiments, the extract or fraction thereof comprises fromabout 1.0% to about 20.0%, from about 2.0% to about 15.0%, from about2.5% to about 6.5%, from about 8% to about 12.0% phenolic acid. In someembodiments, the phenolic acid is selected from the group consisting ofchlorogenic acid, cafeic acid, ferulic acid, isoferulic acid andcombinations thereof. In some embodiments, chlorogenic acid represents amajor proportion of phenolic acid in the composition, for example,greater than 85%, greater than 90%, greater than 95%.

In some embodiments, the extract or fraction thereof comprises fromabout 1.0% to about 20.0%, from about 2.0% to about 15.0%, from about2.5% to about 6.5%, from about 8% to about 12.0%, about 4% or about 10%chlorogenic acid.

In some embodiments, the extract or fraction thereof comprises fromabout 1.0% to about 20.0%, from about 1.0% to about 15.0%, from about2.5% to about 6.5%, from about 8.0% to about 12.0% flavan-3-ol. In someembodiments, the flavan-3-ol is selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof.

In some embodiments, the extract or fraction thereof comprises fromabout 1.0% to about 20.0%, from about 1.0% to about 15.0%, from about2.5% to about 10.0%, from about 2.5% to about 6.5%, from about 8.0% toabout 10.0% epicatechin.

In some embodiments, the fraction has the following phenolic profile:from about 60.0% to about 95.0% flavonol selected from the groupconsisting of quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside and combinationsthereof; from about 0.5% to about 10.0% cyanidin-3-O-galactoside; fromabout 1.0% to about 10.0% dihydrochalcone selected from the groupconsisting of phloridzin, phloritin and combinations thereof; from about1.0% to about 20.0% phenolic acid selected from the group consisting ofchlorogenic acid, cafeic acid, ferulic acid, isoferulic acid andcombinations thereof; and from about 1.0% to about 20.0% flavan-3-olselected from the group consisting of epigallocatechin, catechin,epicatechin and combinations thereof, wherein the percentages are basedthe total weight of phenolic content of the fraction and wherein thetotal does not exceed 100%.

In one embodiment, the fraction has the following phenolic profile: fromabout 70.0% to about 90.0% flavonol selected from the group consistingof quercetin, Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside,Q-3-O-glucoside, Q-3-O-rhamnoside and combinations thereof; from about0.5% to about 5.0% cyanidin-3-O-galactoside; from about 2.0% to about10.0% dihydrochalcone selected from the group consisting of phloridzin,phloritin and combinations thereof from about 2.0% to about 15.0%phenolic acid selected from the group consisting of chlorogenic acid,cafeic acid, ferulic acid, isoferulic acid and combinations thereof andfrom about 1.0% to about 15.0% flavan-3-ol selected from the groupconsisting of epigallocatechin, catechin, epicatechin and combinationsthereof.

In one embodiment, the fraction comprises: from about 80.0% to about90.0% flavonol selected from the group consisting of quercetin,Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside,Q-3-O-rhamnoside and combinations thereof from about 0.5% to about 2.5%cyanidin-3-O-galactoside; from about 3.5% to about 7.5% dihydrochalconeselected from the group consisting of phloridzin, phloritin andcombinations thereof from about 2.5% to about 6.5% phenolic acidselected from the group consisting of chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid and combinations thereof and from about2.5% to about 6.5% flavan-3-ol selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof. Thiswould, for example, encompass fraction F5 in Table 2a.

In one embodiment, the fraction comprises: from about 70.0% to about80.0% flavonol selected from the group consisting of quercetin,Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside,Q-3-O-rhamnoside and combinations thereof from about 2.0% to about 6.0%cyanidin-3-O-galactoside; from about 1.5% to about 4.5% dihydrochalconeselected from the group consisting of phloridzin, phloritin andcombinations thereof from about 8.0% to about 12.0% phenolic acidselected from the group consisting of chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid and combinations thereof and from about8.0% to about 12.0% flavan-3-ol selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof. Thiswould, for example, encompass fraction F4 of Table 2a.

In some embodiments, the extract or fraction thereof comprises: fromabout 10.0% to about 60.0%, from about 15.0% to about 50.0%, from about20.0% to about 50.0%, from about 20.0% to about 35.0%, from about 20.0%to about 30.0%, from about 20.0% to about 25.0% Q-3-O-galactoside; fromabout 10.0% to about 60.0%, from about 15.0% to about 50.0%, from about20.0% to about 50.0%, from about 20.0% to about 40.0%, from about 20.0%to about 35.0%, from about 20.0% to about 30.0%, from about 20.0% toabout 25.0%, from about 30.0% to about 35.0% Q-3-O-rhamnoside; fromabout 1.0% to about 20.0%, from about 5.0% to about 15.0%, from about7.0% to about 13.0%, from about 5.0% to about 10.0%, from about 10.0% toabout 15.0%, Q-3-O-rutinoside; from about 1.0% to about 20.0%, fromabout 5.0% to about 15.0%, from about 7.0% to about 13.0%, from about10.0% to about 15.0%, from about 10.0% to about 15.0%, Q-3-O-glucoside;from about 0.5% to about 10.0%, from about 0.5% to about 5.0%, fromabout 0.5% to about 2.5, from about 0.5% to about 2.0%, from about 1.0%to about 2.0%, from about 2.0% to about 6.0%, from about 3.5% to about5.5%, from about 4.0% to about 5.0% cyanidin-3-O-galactoside; from about0.5% to about 10.0%, from about 1.0% to about 10.0%, from about 2.0% toabout 10.0%, from about 1.0% to about 5.0%, from about 1.5% to about4.5%, from about 3.5% to about 7.5%, about 3.0% to about 6.0%, about 3%,about 5% phloridzin; from about 1.0% to about 20.0%, from about 2.0% toabout 15.0%, about 5.0% to about 15.0%, from about 2.5% to about 6.5%,from about 8% to about 12.0%, about 4% or about 10% chlorogenic acid;and from about 1.0% to about 20.0%, from about 1.0% to about 15.0%, fromabout 5.0% to about 15.0%, from about 2.5% to about 10.0%, from about2.5% to about 6.5%, from about 5.0% to about 10.0%, from about 8.0% toabout 10.0% epicatechin.

In some embodiments, the extract or fraction thereof comprises: fromabout 20.0% to about 30.0% Q-3-O-galactoside; from about 20.0% to about30.0% Q-3-O-rhamnoside; from about 10.0% to about 15.0%,Q-3-O-rutinoside; from about 10.0% to about 15.0%, Q-3-O-glucoside; fromabout 2.0% to about 6.0% cyanidin-3-O-galactoside; from about 1.0% toabout 5.0%, phloridzin; from about 8% to about 12.0% chlorogenic acid;and from about 5.0% to about 10.0% epicatechin.

In some embodiments, the extract or fraction thereof comprises: fromabout 20.0% to about 35.0% Q-3-O-galactoside; from about 20.0% to about35.0% Q-3-O-rhamnoside; from about 5.0% to about 15.0% Q-3-O-rutinoside;from about 5.0% to about 15.0% Q-3-O-glucoside; from about 0.5% to about2.5 cyanidin-3-O-galactoside; from about 1.0% to about 5.0% phloridzin;from about 5.0% to about 15.0% chlorogenic acid; and from about 5.0% toabout 10.0% epicatechin.

The percentages of phenolic compounds in the extract or fraction thereofare based the total weight of monomeric phenolic compounds quantified inthe extract or fraction thereof and do not exceed 100%.

In some embodiments, the phenolic content comprises from about 5000 toabout 15000 mg/L flavonol selected from the group consisting ofquercetin, Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside,Q-3-O-glucoside, and Q-3-O-rhamnoside; from about 1 to about 1000 mg/Lcyanidin-3-O-galactoside; from about 100 to about 1000 mg/Ldihydrochalcone selected from the group consisting of phloridzin andphloritin; from about 100 to about 2000 mg/L phenolic acid selected fromthe group consisting of chlorogenic acid, cafeic acid, ferulic acid andisoferulic acid; and from about 100 to about 2000 mg/L flavan-3-olselected from the group consisting of epigallocatechin, catechin andepicatechin. This would include, for example, various compositions madeaccording to the method disclosed in Example 1. Amounts are based on anoriginal elution volume of 800 mL as described in Example 1.

In some embodiments, the phenolic content comprises from about 8000 toabout 13000 mg/L flavonol selected from the group consisting ofquercetin, Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside,Q-3-O-glucoside, and Q-3-O-rhamnoside; from about 80 to about 700 mg/Lcyanidin-3-O-galactoside; from about 300 to about 900 mg/Ldihydrochalcone selected from the group consisting of phloridzin andphloritin; from about 400 to about 1600 mg/L phenolic acid selected fromthe group consisting of chlorogenic acid, cafeic acid, ferulic acid andisoferulic acid; and from about 500 to about 1500 mg/L flavan-3-olselected from the group consisting of epigallocatechin, catechin andepicatechin.

In some embodiments, the phenolic content comprises from about 10000 toabout 12000 mg/L flavonol selected from the group consisting ofquercetin, Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside,Q-3-O-glucoside, and Q-3-O-rhamnoside; from about 100 to about 200 mg/Lcyanidin-3-O-galactoside; from about 700 to about 800 mg/Ldihydrochalcone selected from the group consisting of phloridzin andphloritin; from about 500 to about 600 mg/L phenolic acid selected fromthe group consisting of chlorogenic acid, cafeic acid, ferulic acid andisoferulic acid; and from about 600 to about 700 mg/L flavan-3-olsselected from the group consisting of epigallocatechin, catechin andepicatechin.

Mass spectrometry analysis indicates that the major flavonoid componentsof exemplary fractions F4 and F5 are quercetin-3-O-glucoside,quercetin-3-O-galactoside, quercetin-3-O-rhamnoside,quercetin-3-O-rutinoside, epicatechin, chlorogenic acid and phloridzin.

In some embodiments, the phenolic content comprises from about 80.0% toabout 90.0% flavonol selected from the group consisting ofQ-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnosideand combinations thereof; from about 1.0% to about 2.0%cyanidin-3-O-galactoside; from about 4.5% to about 6.5% phloridzin; fromabout 2.5% to about 4.5% chlorogenic acid; and from about 2.5% to about5.5% epicatechin. In some embodiments, this would include, for example,Fraction F5 of Table 2a.

In one embodiment, the fraction comprises around 70.0% to about 80.0%flavonol selected from the group consisting of Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside and combinationsthereof; about 4.0% to about 6.0% cyanidin-3-O-galactoside; from about2.0% to about 4.0% phloridzin; from about 8.0% to about 12.0%chlorogenic acid; and from about 6.0% to about 10.0% epicatechin. Insome embodiments, this would include, for example, Fraction F4 of Table2a.

In some embodiments the, composition further comprises one or moreexcipients, preferably physiologically acceptable or pharmaceuticallyacceptable excipients. Suitable excipients are known to those of skillin the art and will be dependent on the dosage from being made, e.g. anatural health product versus a pharmaceutical. It will be understoodthat other active ingredients may be included in the composition, suchas food ingredients and other medicines.

The extract or fraction thereof may be present in a composition ordosage form in any suitable amount that will achieve the desiredtherapeutic, prophylactic, or health-promoting effect. For instance, theextract or fraction thereof may be present in an amount of about 0.1 wt% to about 99.9 wt %, about 1 wt % to about 85 wt %, about 1 wt % toabout 60 wt %, about 1 wt % to about 50 wt %, about 1 wt % to about 40wt %, about 1 wt % to about 30 wt %, about 5 wt % to about 65 wt %,about 5 wt % to about 30 wt %, about 10 wt % to about 30 wt % of thecomposition.

In another aspect, there is provided a process for extracting phenoliccompounds from apple skins to provide a crude extract and furtherfractionating the crude extract to provide compositions having uniquephenolic profiles.

The phenolic extract is obtainable by an aqueous extraction processhaving the following steps: obtaining a sample of apple skins; treatingthe skins to inhibit degradation of phenolic compounds; optionallydehydrating the skins and converting the skins to a powder form;extracting the skins one or more times with a food-grade solvent, suchas, ethanol; optionally subjecting the skins to sonication duringextraction; removing solids to obtain a phenolic extract; optionallyconcentrating the phenolic extract; optionally removing sugars from thephenolic extract, and optionally concentrating, drying and/or freezingthe phenolic extract. From such a process, a crude extract may beobtained.

In some embodiments, the crude phenolic extract is purified, forexample, using column chromatography, for example, chromatography usinga C18 column with ethanol, e.g. 80-100% ethanol, as an eluent. Othereluents may also be sued.

One or more fractions may be obtainable by fractionation of the phenolicextract in a chromatography column using a suitable eluent, or othersuitable method, followed by selection of a fraction having a highphenolic content, in particular, a high flavonol content. Thus, in someembodiments, the process further comprises subjecting the crude extractto fractionation to obtain eluted fractions; and optionallyconcentrating, drying and/or freezing the fractions.

In some embodiments, the eluent is ethanol and the chromatography isflash chromatography with a C18 column using a polymeric sorbent.

In some embodiments, fractions having a high phenolic content, inparticular, a high flavonol content, are eluted in about 40% to about60% ethanol. In one embodiment, the fraction is eluted in about 45% toabout 50% ethanol. In one embodiment, the fraction is eluted in about45% ethanol, such as, for example, fraction F4 in Table 2a. In oneembodiment, the fraction is eluted in about 50% ethanol, such as, forexample, fraction F5 in Table 2a.

The step of treating the skins to inhibit degradation of phenoliccompounds may also be carried out by any suitable method, e.g. blanchingor salt treatment.

In some embodiments, the step of treating the skins to inhibitdegradation of phenolic compounds is carried out by treating the skins(whether dehydrated or not) in a salt solution, for example 1 to 10%calcium chloride (CaCl₂) solution, or 1% to 5% CaCl₂, e.g. 2% CaCl₂,shortly after peeling the fruit, for example, within 10 minutes ofpeeling, to thereby preserve the antioxidant compounds present in theskins. In some embodiments, the skins are soaked in a salt solution at atemperature of about 40° C. to about 100° C., 50° C. to about 70° C., inparticular at about 55° C., for about 5 to about 60 minutes, e.g. 10minutes.

The peels soaked in salt solution may be extracted directly orfreeze-dried for storage and/or transport.

The skins may be ground into a slurry, for example, using an UrsherMill. In some embodiments, the peels are dehydrated. For example, saltsoaked apple peels may be dried in an oven with air circulation at atemperature of about 50° C. to about 70° C., in particular at about60±2° C., for at least about 24 hours, or about 24 to 72 hours, e.g.about 48 hours.

In some embodiments, dehydrated peels are converted into a fine powder,for example, using mechanical grinding means, such as a coffee grinderor an industrial equivalent. The powdered skins may be used directly ormay be frozen for later use.

The skins are extracted using a food-grade solvent. Any suitable solventmay be used, including but not limited to, 40% to 100% methanol orethanol, e.g. 95% to 100%. In some embodiments, the solvent is 95% to100% ethanol.

The extraction process is preferably aided by sonication or similarmechanism for disrupting the skins. The conditions to extract thephenolic compounds into the solvent may comprise sonicating for asufficient period of time, for example, about 5 minutes to 2 hours, orabout 10 minutes to about 30 minutes, e.g. 10 or 15 minutes, to releasethe phenolic compounds into the solvent. An ultrasonication bath or thelike may be used.

The extraction step may be repeated as desired. In some embodiments, theextraction step is preformed twice.

Following extraction, solids may be removed by any suitable method, forexample, centrifugation or filtration.

The crude extract may optionally be concentrated, for example, to about1% to about 50%, to about 5% to about 20%, e.g. about 10%, originalvolume. In some embodiments, the crude extract may be reduced to drynessto provide a solid concentrate, which may be later reconstituted in asuitable medium for use or for fractionation.

The crude extract comprising phenolic compounds may optionally betreated under conditions to remove sugar compounds. The sugars may beremoved, for example, by chromatography, for example, flash columnchromatography. The column may, for example, be flushed with water oneor more times, e.g. two or three times, to remove the sugars. In anotherembodiment, the solid support or stationary phase in the column is a C₁₈resin or any other support that absorbs hydrophobic compounds (forexample, Amberlite XAD 16 or Sorbent SP207-05). The removal of sugarfrom the crude extract may be monitored, for example, by measuring theBrix value of wash water using a refractometer. In some embodiments, theBrix value is monitored until it reaches less than 5%, e.g. less than1%, and then the washing step is terminated.

The crude extract comprising phenolic compounds may optionally betreated under conditions to remove lipids, carontenoids, chlorophyllsand/or proanthocyanidins (suitable techniques are described, forexample, in Huber and Ruphasinghe, 2009).

The crude extract may then be subjected to fractionation to obtaineluted fractions having unique combinations of phenolic compounds, orunique phenolic profiles. The fractions may also contain other agentsbesides the phenolic compounds. The methods, reagents and conditionsemployed can affect the constitution of the individual fractions and,once a desirable fraction is obtained, should be controlled forconsistency from batch to batch.

Fractionation may be performed, for example, using flash chromatographyusing a polymeric sorbent. One example of a suitable polymeric sorbentis Sorbent SP207-05 Sepabeads resin brominated styrenic adsorbent;particle size 250 μm, surface area 630 m²/g. In some embodiments, thecolumn contains about 500 to about 800 g, e.g. 600 g, sorbent.

The phenolic compounds may be eluted using any suitable solvent andelution protocol. In some embodiments, the phenolic compounds are elutedusing a lower alcohol, such as, methanol, ethanol or propanol. In someembodiments, the eluent is a food-grade lower alcohol. In someembodiments, the phenolic compounds are eluted using a step gradient ofethanol (see Table 1). The eluted fractions may optionally be dried,diluted or concentrated. The eluted fractions may be concentrated, forexample, using evaporation techniques, such as rotary evaporation at,for example, 20° C. to 60° C., e.g. 45° C. The eluted fractions may beevaporated to about less than 5%, e.g. about 2.5%, original elutionvolume. The concentrated or dried samples may later be resuspended in asuitable medium, such as water or other aqueous medium.

In some embodiments, the process of preparation comprises: obtaining asample of apple skins; treating the skins with a CaCl₂ solution shortlyafter (e.g. within 10 minutes) peeling to inhibit degradation ofphenolic compounds; dehydrating the skins and grinding the dried skinsto a powder; extracting the powder at least twice with ethanol andsubjecting the suspension to sonication during each extraction step;removing solids by centrifugation or equivalent method to obtain a crudephenolic extract; concentrating the crude extract to about 10% originalvolume; removing sugars from the concentrated crude extract until theBrix value is less than about 1%; subjecting the crude extract tostep-gradient fractionation with ethanol on a C-18 chromatography columnaccording to the schedule below to obtain eluted fractions:

Fraction Number Percent ethanol F1 20% F2 30% F3 40% F4 45% F5 50% F655% F7 60% F8 65% F9 70% F10 75% F11 80% F12 90% F13 100%  F14 100%  F15100% selecting eluted fractions F4 and/or F5; and optionally concentrating ordrying the selected fractions prior to use. In some embodiments, afraction having a high phenolic content is selected. In someembodiments, a fraction having a high flavonol content is selected, inparticular high quercetin glycosides.

In some embodiments, F4 is selected.

In some embodiments, F5 is selected.

In some embodiments, the treating step involves subjecting the skins toa solution of CaCl₂ in water (w/v) at about 55° C. for about 10 minutes.The solution of CaCl₂ may for example be about 1%-about 10%, about1%-about 5%, or about 2% CaCl₂.

In some embodiments, the skins are dried at about 60° C.±2° C., forabout 48 h, or until dry, prior to being ground to a powder.

In some embodiments, the extraction step is carried out by sonicatingthe powder in a ratio of 200 g powder to 1 L ethanol two times for 15minutes with 10 minute interval resulting in a crude extract having aratio of 200 g powder to 2 L ethanol.

In some embodiments, the crude extract is concentrated using a rotaryevaporation system until the original volume is reduced to about 10%.

In some embodiments, the fractionation is carried out using flashchromatography using a polymeric sorbent.

In some embodiments, the sugars are removed by washing the column with 2to 3 times bed volume of water while monitoring the Brix value of thewater exiting the column.

In some embodiments, the step-gradient fractionation is carried out with800 ml ethanol solution for each fraction.

In some embodiments, the selected fractions are concentrated to about2.5% of the original elution volume.

In some embodiments, the composition comprises eluted fraction F5 whichmay optionally be diluted, dried or concentrated.

In some embodiments, the composition comprises eluted fraction F4 whichmay optionally be diluted, dried or concentrated.

In another aspect, there is provided a dietary supplement or naturalhealth product for reducing or preventing oxidative stress comprising aphenolic composition as described herein. In some embodiments, thedietary supplement or natural health product is in the form of aconcentrate, a liquid, a powder, an emulsion, a suspension, a film, apaste, a gel, a gum, a drop, a tablet, a capsule, a microcapsule or afood additive.

There is also provided a dietary supplement or natural health productfor preventing or reducing damage due to oxidative stress comprising afraction as described herein.

In another aspect, there is provided a functional food or beveragecomprising a phenolic composition as described herein. Variousfunctional food and beverage formats are known in the industry.

In another aspect, there is provided a method of preventing or treatinga disease or condition associated with oxidative stress and/orinflammation, comprising administering to a subject an effective amountof a composition as described herein comprising a phenolic extract orfraction thereof derived from apple skin.

Compositions described herein were shown to reduce neuron death due tooxygen glucose deprivation in vitro and to reduce brain injury anddisability in vivo in animal models of diseases and conditions mediatedby oxidative stress and inflammation, including neuroinflammation,excitotoxicity, apoptosis, necrosis and/or autoimmunity, such as strokeand multiple sclerosis. The compositions disclosed herein are thereforebelieved to be useful in preventing or treating a disease or conditionassociated with oxidative stress and/or inflammation, includingneuroinflammation, excitotoxicity, apoptosis, necrosis and/orautoimmunity. The compositions disclosed herein are also believed to beuseful in preventing or treating neuron cell death due to oxidativestress, neuroinflammation, excitotoxicity, apoptosis, necrosis and/orautoimmunity.

Without wishing to be bound by theory, at least the following can benoted from the examples. With respect to the oxygen-glucose deprivation(OGD) experiment on cortical neurons, glutamate antagonists are known tobe protective. If therefore possible that F4 reduced the detrimentaleffects of OGD by preventing excitotoxicity and oxidative stress,perhaps by increasing resistance to this cause of neuronal cell death bystabilizing calcium overload. With respect to EAE (animal model of MS),neuroinflammation resulting from autoimmune mechanism is the primarycause of paralysis, thus F4 may be blocking this type of injury. Inhypoxia-ischemia (HI)-induced brain injury, neuroinflammation,excitotoxicity, as well oxidative stress are all at play. Thecompositions disclosed herein were also shown to inhibitphosphodiesterase IV and inflammatory cytokine production. Thecompositions disclosed herein may therefore also improve cognition byreducing neuroinflammation and oxidative stress as well as blockingphosphodiesterase IV, increasing levels of cAMP necessary for memoryconsolidation.

In some embodiments, the disease or condition associated with oxidativestress and/or inflammation includes, but is not limited to, aging,chronic fatigue syndrome, neurodegenerative disorders, autoimmunedisorders, metabolic disorders, and vascular disorders.Neurodegenerative disorders include, for example, Parkinson's disease,Alzheimer's disease, retinal degeneration, hearing loss, fragile Xsyndrome, traumatic brain injury, spinal cord injury, head injury anddemyelinating disorders. Demyelinating disorders include, for example,multiple sclerosis, devic's, progressive multifocal leukoencephalopathy,optic neuritis, leukodystrophies, charcot-marie tooth, andguillian-barre syndrome. Autoimmune disorders include, for example, typeI diabetes, rheumatoid arthritis, lupus, colitis and Crohn's disease.Vascular disorders include, for example, stroke, atherosclerosis,myocardial infarction and vascular dementia. Beneficial effects offlavonoids may derive, at least in part, from protection of the cerebralvasculature and microvasculature, such as in stroke, MS, and vasculardementia. Metabolic disorders include, for example, obesity, type IIdiabetes, and lipid disorders, such as, hypercholesteremia. Thecompositions described herein may also be useful in the treatment orprevention of certain oxidative stress-mediated orphan disorders, suchas amyotrophic lateral sclerosis (ALS), primary progressive MS,charcot-marie-tooth disease, and spinal muscular atrophy. It will beunderstood that diseases and conditions associated with oxidative stressand/or inflammation may fall into more than one category listed above.

In one embodiment, the disease or condition is a vascular disease, suchas stroke.

In some embodiments, the disease or condition is a neurodegenerativedisease, such as multiple sclerosis, Parkinson's disease, or Alzheimer'sdisease.

The composition may be administered in any suitable dosage form. Thedosage form may be administered in an amount to deliver an effectiveamount of the extract or faction thereof. The effective amount may be atherapeutically effective amount, a prophylactically effective amount oran amount for general promotion of good heath.

The dosage requirements vary with the particular formulations and dosageforms employed, the route of administration, the severity of thesymptoms presented and the particular subject being treated. Treatmentwill generally be initiated with small dosages less than the optimumdose of the compound. Thereafter the dosage is increased until theoptimum effect under the circumstances is reached. Precise therapeuticdosages may be determined by the administering physician based onexperience with the individual subject treated. In general, the activeagent is most desirably administered at a concentration that willgenerally afford effective results without causing harmful ordeleterious side effects, and can be administered either as a singleunit dose, or if desired, the dosage may be divided into convenientsubunits at suitable times throughout the day.

In addition, in vitro or in vivo assays may optionally be employed tohelp identify optimal dosage ranges. For example, a dose may beformulated in animal models to achieve a beneficial circulatingcomposition concentration range. Initial doses may also be estimatedfrom in vivo data, e.g., animal models, using techniques that are knownin the art. Such information may be used to more accurately determineuseful doses in humans. One having ordinary skill in the art mayoptimize administration to humans based on animal data.

The amount of phenolic compounds administered can depend on, among otherfactors, the subject, the weight of the subject, the health of thesubject, the disease being treated, the severity of the affliction, theroute of administration, the potency of the active agent, and thejudgment of the prescribing physician.

The amount of active agent that will be effective in the treatment of aparticular disease, disorder, or condition disclosed herein will dependon the nature of the disease, disorder, or condition, and can bedetermined by standard clinical techniques known in the art.

In some embodiments, the compositions described herein may beadministered in multiple doses. In some embodiments, the compositionsdescribed herein may be administered over multiple days, e.g. 2, 3, 4,5, 7, 10, 15, 20, 30, 40, or more days. The examples provided hereinsuggest that the protective effects of the compositions may increaseover multiple doses (as for example, with antidepressants), thusprolonged or chronic administration may be preferred in some cases.Without being bound by theory, the fact that better results are seenwith multiple doses may indicate delayed downstream effects, forexample, effects on gene transcription.

In some cases, prolonged administration may be advised in subjectssusceptible to one or more of the diseases or conditions recited herein,for example, aging populations or populations with geneticpredisposition to certain diseases or conditions (e.g. Parkinson's,Alzheimer's or MS), or subjects having previously experienced a symptomor event associated with one or more of the diseases or conditionsrecited herein (e.g. stroke).

In some embodiments, the method comprises daily administration of acomposition as disclosed herein to a subject. In some embodiments, themethod comprises twice daily administration of a composition asdisclosed herein to a subject. In some embodiments, the method comprisesthrice daily administration of a composition as disclosed herein to asubject.

The administered dose is less than a toxic dose. Toxicity of thecompositions described herein may be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD50 (the dose lethal to 50% of the population)or the LD100 (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Incertain embodiments, a pharmaceutical composition may exhibit a hightherapeutic index. The data obtained from these cell culture assays andanimal studies may be used in formulating a dosage range that is nottoxic for use in humans.

Studies have shown that administration of quercetin at doses up to 3000mg/kg/day for 28 days were well tolerated in mice (Rutz, M. J. et al2009). In humans, 1 g per day of quercetin for 1 month is safe (Gugler Ret al. 1975). Thus, it is likely that high doses of the compositionsdisclosed herein could similarly be tolerated.

During treatment a dose and dosing schedule may provide sufficient orsteady state systemic concentrations of a therapeutically effectiveamount of flavonoids to prevent or treat a disease or condition. Incertain embodiments, an escalating dose may be administered.

The composition may be administered at intervals for as long asnecessary to obtain an intended or desired effect.

The phenolic compositions described herein may be used on their own fortheir health-promoting benefits, or may be used in conjunction withother medicines. In some cases, it may be possible to reduce the amountof the medicine required, thereby having potential to decrease the costor side effects of treatment.

In another aspect, there is provided a pharmaceutical compositioncomprising a phenolic composition as described herein together with apharmaceutically acceptable excipient, such as a carrier or diluent.Typically, in a pharmaceutical application, the composition isadministered for treatment or prevention of a disease or condition. Insome embodiments, the pharmaceutical composition described herein is foruse in the treatment and/or prevention of a disease or conditionassociated with oxidative stress or inflammation, as described above.The pharmaceutical composition comprises at least one excipient,preferably a pharmaceutical grade excipient, and may be formulated inany suitable dosage form.

The pharmaceutical composition may optionally comprise additional activeingredients, such as a drug.

In some embodiments, the pharmaceutical composition is formulated forenteral administration, topical administration, parenteraladministration, or nasal administration. Enteral administration maycomprise, for example, oral administration.

Parenteral administration may comprise, for example, intravenous,intrarterial, intracerebral, intraperitoneal, intramuscular,subcutaneous, intracardiac, or intraosseous administration. In someembodiments, the parenteral administration is intravenousadministration.

In another aspect, there is provided a use of a composition as describedherein in the preparation of a medicament for the treatment and/orprevention of a disease or condition associated with oxidative stress orinflammation.

In another aspect, there is provided a use of a composition as describedherein for the treatment and/or prevention of a disease or conditionassociated with oxidative stress or inflammation.

In some embodiments, the disease or condition associated with oxidativestress or inflammation.

In another aspect, a phenolic composition as described herein isutilized as a food additive (or food ingredient), and may be addedvarious and diverse foods to provide the food item with a significantquantity of phenolics derived from apple skin. While the phenoliccomposition may be added for its health-promoting benefits, in someembodiments, the phenolic composition may also inhibit or prevent theoxidation of oxidizable compounds, such as polyunsaturated fatty acids(PUFA) and/or lipids, in the food item. The food item may be, forexample, a solid, semi-solid (e.g. pudding, yoghurt), or liquid (e.g.beverage) food item. The food item may, for example, be for human oranimal consumption.

In another aspect, there is provided a cosmetic product comprising aphenolic composition as described herein. The phenolic composition maybe added for its beneficial antioxidant properties and may also inhibitor prevent the oxidation of oxidizable compounds, such aspolyunsaturated fatty acids (PUFA) and/or lipids, in the cosmeticcomposition. The cosmetic product may, for example, be a cream, gel,paste, lotion, emulsion, ointment, or a microencapsulated product.

In another embodiment, there is provided a functional food or beveragecomprising a fraction as disclosed herein.

In other aspects, there are provided commercial packages comprisingcompositions as described herein together with instructions for use.

Any suitable apple cultivar may be used in accordance with the presentdisclosure.

In some embodiments, the apple is a dessert and dual purpose apple, suchas, Adams Pearmain, Alkmene, Ambrosia, Antonovka, Arlet, Ariane,Arkansas Black, Ashmead's Kernel, Aurora Golden Gala, Baldwin, BenDavis, Blenheim Orange, Beauty of Bath, Belle de Boskoop, Bohemia,Braeburn, Brina, Cameo, Clivia, Cornish Gilliflower, Cortland, Cox'sOrange Pippin, Cripps Pink (Pink Lady), Delbarestivale® delcorf,Delbardivine® delfloga, Discovery, Ecolette, Egremont Russet, Elstar,Empire, Esopus Spitzenburg, Fuji, Gala, Ginger Gold, Golden Orange,Golden Delicious, Granny Smith, Gravenstein, Grimes Golden, Haralson,Honeycrisp, Idared, James Grieve, Jazz, Jersey Black, Jonagold,Jonathan, Junaluska, Karmijn de Sonnaville, Knobbed Russet, Liberty,Macoun, McIntosh, Mutsu, Newtown Pippin, Nickajack, Nicola, Novaspy,Novamac, Paula Red, Pink Pearl, Pinova, Rajka, Ralls Genet, Rambo, RedDelicious, Rhode Island Greening, Ribston Pippin, Rome, Royal Gala,Roxbury Russet, Rubens (Civni), Santana, Saturn, Sekai Ichi, Spartan,Stayman, Sturmer Pippin, Summerfree, Taliaferro, Topaz, WorcesterPearmain, York Imperial or Zestar.

In some embodiments, the apple is a cooking apple, for example, selectedfrom Bramley, Calville Blanc d′ hiver, Chelmsford Wonder, Flower ofKent, Golden Noble, Norfolk Biffin or Northern Spy.

In some embodiments, the apple is a cider apple, for example, selectedfrom Brown Snout, Dabinett, Foxwhelp, Harrison Cider Apple, KingstonBlack, Redstreak or Styre.

In some embodiments, the apple is a crabapple.

In some embodiments, the apple is Northern Spy.

The following non-limiting examples are provided to illustrate, andassist the reader in understanding, the present disclosure.

EXAMPLES Example 1 Preparation of Flavonoid-Rich Fractions of Apple SkinExtract

The apple skins of the apple cultivar, Northern Spy, were collected froma commercial pie manufacturer, Apple Valley Foods Inc., Kentville, NS,Canada. Immediately after peeling, the skins were treated with 2% CaCl₂in water (w/v) at 55±5° C. for 10 min to prevent degradation of phenoliccompounds. After draining the excess water and within 3 h of the CaCl₂treatment, the apple skins were transported in plastic containers to theNova Scotia Agricultural College (NSAC). The apple skins were dried inclean plastic trays at 60±2° C. for 48 h using a convection oven withair circulation (Milner Agincourt, ON, Canada). The dried skins wereground into a fine powder using a Willey mill with 1 mm sieve screen(Model Laboratory Heavy Duty, Arthur Thomas Co., Philadelphia, Pa.) andkept in a freezer (−80° C.) for later use. One hundred grams of appleskin powder was weighed into a 2 L flask and sonicated using 1 L ofabsolute ethanol two times for 15 min with 10 min interval. Thesuspension was then transferred into 50 mL corning tubes forcentrifugation at 3000 rpm for 15 min. The supernatants of two of theabove extractions (total of 200 g of apple skins in 2 L of ethanol) werecollected and evaporated to produce 200 mL concentrate using a rotaryevaporation system at 45° C. (Rotavap® R-200, Buchi, Flawil,Switzerland).

For the fractionation of the above concentrated apple skin extract,flash chromatography using a polymeric sorbent (Sorbent SP207-05Sepabeads resin brominated styrenic adsorbent; particle size 250 μm,surface area 630 m²/g) was used. The chromatography column (3.8×45 cm,Sati International Scientific Inc., Dorval, QC, Canada) containing the600 g adsorbent was conditioned with deionized water and loaded withapple skin extract at the top of the column. The column was immediatelywashed with water by sending 2 to 3 times of bed volume of water throughit. The removal of sugar from the crude extract was monitored bymeasuring the Brix value of wash water using a refractometer. Once theBrix value was less than 1%, washing step was terminated. The phenoliccompounds retained in the column were eluted using a step gradient ofethanol (Table 1, 800 mL of each elusion) and the elute was concentratedto 20 mL using a rotary evaporator (Rotavap® R-200, Buchi, Flawil,Switzerland) at 45° C.

TABLE 1 The percentage of ethanol used for the step-gradient of elusionsof phenolic compounds using the C-18 column. Fraction Number Percentethanol F1 20% F2 30% F3 40% F4 45% F5 50% F6 55% F7 60% F8 65% F9 70%F10 75% F11 80% F12 90% F13 100%  F14 100%  F15 100% 

LC-MS/NIS Analysis of Phenolics in the C-18 Fractions.

Analyses of major individual phenolic compounds present in 15 apple peelfractions (Tables 2a and 2b) were performed according to the procedurereported by Rupasinghe et al. (2010). The apple peel fractions arenumbered F1 through F15.

All analyses were performed using a Waters Alliance 2695 separationsmodule (Waters, Milford, Mass.) coupled with a Micromass Quattro microAPI MS/MS system and controlled with Masslynx V4.0 data analysis system(Micromass, Cary, N.C.). The column used was a Phenomenex Luna C₁₈ (150mm×2.1 mm, 5 μm) with a Waters X-Terra MS C₁₈ guard column. For theseparation of the flavonol, flavan-3-ol, phenolic acid anddihydrochalcone compounds, a gradient elution was carried out with 0.1%formic acid in water (solvent A) and 0.1% formic acid in acetonitrile(solvent B) at a flow rate of 0.35 mL/min. A linear gradient profile wasused with the following proportions of solvent A applied at time t(min); (t, A %): (0, 94%), (9, 83.5%), (11.5, 83%), (14, 82.5%), (16,82.5%), (18, 81.5%), (21, 80%), (29, 0%), (31, 94%), (40, 94%). Theanalysis of cyanidin-3-β-galactoside was carried out using the mobilephases of 5% formic acid in water (solvent A) and 5% formic acid inmethanol (solvent B) at a flow rate of 0.35 mL/min. The linear gradientprofile used was as follows; (t, A %): (0, 90%), (10, 70%), (17, 60%),(21, 48.8%), (26, 36%), (30, 10%), (31, 90%), (37, 90%).

Electrospray ionization in negative ion mode (ESI−) was used for theanalysis of the flavonol, flavan-3-ol, phenolic acid and dihydrochalconecompounds. The following conditions were used: capillary voltage −3000V, nebulizer gas (N₂) temperature 375° C. at a flow rate of 0.35 mL/min.For the analysis of cyanidin-3-O-galactoside, electrospray ionization inpositive ion mode (ESI+) was used. The settings for the positive ionexperiments were as follows: capillary voltage 3500 V, nebulizer gas375° C. at a flow rate of 0.35 mL/min. The cone voltage (25 to 50 V) wasoptimized for each individual compound. Multiple reaction-monitoring(MRM) mode using specific precursor/product ion transitions was employedfor quantification in comparison with standards: m/z 301→105 forQuercetin (Q), m/z 609→301 for Q-3-O-rutinoside, m/z 463→301 forQ-3-O-glucoside and Q-3-O-galactoside, m/z 448→301 for Q-3-O-rhamnoside,m/z 595→301 for Q-3-O-peltoside, m/z 273→167 for phloritin, m/z 435→273for phloridzin, m/z 353→191 for chlorogenic acid, m/z 179→135 for cafeicacid, m/z 193→134 for ferulic acid and isoferulic acid, m/z 449→287 forcyanidin-3-O-galactoside, m/z 289→109 for catechin, m/z 290→109 forepicatechin, and m/z 305→125 for epigalocatechin. In MRM experiments,both quadrupoles were operated at unit resolution.

TABLE 2a Concentration (mg/L) of phenolic compounds in Fractions 1 to 7.Fraction number Phenolic compound F1 F2 F3 F4 F5 F6 F7 Quercetin (Q) 0.10.1 0.6 9.9 20.6 11.9 50.2 Q-3-O-paltoside 0 0 7.2 63.8 29.0 2.3 0Q-3-O-rutinoside 0 0 293.3 1535.7 1105.0 285.1 30.3 Q-3-O-galactoside2.0 1.2 566.8 2914.9 3914.7 2346.1 575.8 Q-3-O-glucoside 0.5 0.3 101.61474.8 1657.1 721.5 101.7 Q-3-O-rhamnoside 1.8 1.0 86.9 2771.6 4339.23112.8 973.6 Total Flavonols 4.4 2.6 1056.40 8770.7 11065.6 6479.71731.6 (48.4%)  (1.6%) (23.1%) (72.3%) (84.2%) (86.9%) (85.7%)Cyanidin-3-O- 0 0 527.6 559.4 167.4 29.2 6.3 galactoside   (0%)   (0%)(11.5%) (4.6%) (1.2%) (0.3%) (0.3%) Phloridzin 1.3 0.9 7.7 386.8 711.5614.1 239.9 Phloritin 0.8 0 0.8 1.0 1.2 1.2 1.8 Total 2.1 0.9 8.5 387.8712.7 615.3 241.7 dihydrochalcone (23.1%) (0.56%) (0.1%) (3.1%) (5.4%)(8.2%) (11.9%) Chlorogenic acid 1.9 99.8 1663.0 1221.1 502.8 97.7 10.4Cafeic acid 0.7 0.9 2.1 43.6 25.1 7.0 1.4 Ferulic acid 0 0 1.2 0 13.320.3 5.8 Isoferulic acid 0 0 0 3.7 23.5 13.9 4.3 Total phenolic acids2.6 100.7 1666.3 1268.4 564.7 138.9 21.9 (28.6%) (62.8%) (36.4%) (10.4%)(4.3%) (1.9%) (1.1%) Epigallocatechin 0 0.9 7.4 0.9 2.9 1.3 0 Catechin 015.7 210.4 106.8 46.0 13.2 1.7 Epicatechin 0 39.4 1104.8 1044.3 579.3178.2 16.8 Total Flavan-3-ol 0 56.0 1322.6 1152.0 628.2 192.7 18.5  (0%) (34.9%) (28.9%) (9.5%) (4.8%) (2.6%) (0.9%) Total phenolics 9.1160.2 4578.4 12138.3 13138.6 7455.8 2020

TABLE 2b Concentration (mg/L) of phenolic compounds in Fractions 8 to15. Fraction number Phenolic compound F8 F9 F10 F11 F12 F13 F14 F15Quercetin (Q) 45.0 53.8 68.2 1.4 32.1 10.7 7.7 0.5 Q-3-O-paltoside 0 0 00 0 0 0 0 Q-3-O-rutinoside 4.9 1.5 0 0 0 0 2.4 0 Q-3-O-galactoside 75.821.7 11.7 6.8 5.5 7.5 33.4 10.6 Q-3-O-glucoside 14.2 3.7 2.4 1.2 1.2 1.25.4 2.1 Q-3-O-rhamnoside 176.4 34.3 11.9 5.7 3.9 3.2 19.4 19.5 Total316.3 115 94.2 15.1 42.7 22.6 68.3 32.7 Flavonols (82.4%) (84.4%)(89.6%) (70.6%) (65.8%) (45.9%) (77.4%) (74.7%) Cyanidin-3-O- 1.9 1.10.8 0.5 0.6 0.6 0.7 0.5 galactoside (0.4%) (0.8%) (0.7%) (2.3%) (0.9%)(1.2%) (0.8%) (1.1%) Phloridzin 53.6 12.0 4.3 2.6 2.2 1.7 6.5 7.9Phloritin 3.1 3.3 1.8 1.0 0.1 0.9 0.9 0.8 Total 56.7 15.3 6.1 3.6 3.22.6 7.4 8.7 dihydrochalcone (14.7%) (11.2%) (5.8%) (16.8%) (4.9%) (5.3%)(8.4%) (19.9%) Chlorogenic acid 3.3 2.3 2.0 1.7 10.5 9.5 4.2 1.2 Cafeicacid 0.8 0 0.7 0 0.8 0.8 0.8 0.7 Ferulic acid 1.7 1.1 0 0 0 0 0 0Isoferulic acid 0.9 0 0 0 0 0 0 0 Total phenolic acids 6.7 3.4 2.7 1.711.3 10.3 5.0 1.9 (1.7%) (2.5%) (2.6%) (7.9%) (17.4%) (20.9%) (5.7%)(4.3%) Epigallocatechin 0 0 0 0 0 0 0 0 Catechin 0 0 0 0 1.9 1.8 0.9 0Epicatechin 2.3 1.5 1.3 0.5 5.2 11.3 5.9 0 Total Flavan-3-ol 2.3 1.5 1.30.5 7.1 13.1 6.8 0 (0.6%) (1.1%) (1.2%) (2.3%) (10.9%) (26.6%) (7.7%)Total phenolics 383.9 136.3 105.1 21.4 64.9 49.2 88.2 43.8

In Tables 2a and 2b, the values shown in brackets represent thepercentage of that component relative to the total phenolic contentmeasured in that fraction, where the total phelolic amount shown at thebottom of each row represents 100% for that fraction. It can be seenthat different fractions may have different phenolic profiles based onthe relative amounts of each component in the fraction. The variousfractions may further contain additional unidentified components.

It was determined that Fractions F4 and F5 had the highest concentrationof phenolic compounds, with F4 having 12138.3 mg/L, and F5 having13138.6 mg/L total phenolics concentration per 800 ml eluted fraction.

When looking at the amount of a particular component (e.g. totalphenolic content) across horizontal rows in Tables 2a and 2b, therelative amount of that component compared to the crude extract can beroughly estimated. For example, F4 and F5 contain approximately 30% and32.5%, respectively, of the total phenolic content of the 15 fractions.Fractions F4 and F5 also had the highest flavonol content. Withoutwishing to be bound by theory, it is believed that these fractions areparticularly effective due to the high flavonol content of thesefractions. It is also possible that there are synergies between thecomponents in the fractions.

Example 2 Neuroprotective Effects of F5 in an Animal Model of Stroke

F5 Treatment

The neuroprotective potential of phenolic composition F5 (Table 2a)derived from apple peel was investigated in an hypoxia-ischemia (HI)model of brain injury. The total phenolic content of the fraction wasmeasured, based on the phenolic compounds listed in Table 2a. Theextract was administered to C57/b16 mice (6-8 weeks old) by oral gavagefor 3 consecutive days at a dose of 50 mg phenolics/kg body weight. Micein a control group were given vehicle (water) at a volume of 0.01 ml/gbody weight by oral gavage for 3 consecutive days. Twenty-four hoursafter the last dose of extract or vehicle, the mice were subjected tohypoxia-ischemia (HI).

A separate study had shown that a single dose (50 mg phenolics/kg) wasnot sufficient to significantly protect against damage caused byhypoxia-ischemia compared to vehicle treatment group (data not shown),suggesting that increased dosage or multiple treatments are required.

Brain Injury Induced by Hypoxia-Ischemia (HI)

The hypoxia-ischemia (HI) method originally reported by Levine (1960)for rats was used to induce cerebral ischemia. The procedure wasslightly modified to accommodate the use of adult mice. Mice wereanaesthetised using Isoflurane in an induction chamber (3% vaporisedwith medical oxygen at a rate flow of 3 L/min). Anaesthesia wasmaintained with 2% Isoflurane vaporised with oxygen flowing at a rate of1.5 L/min. A small midline incision was made on the ventral neck withscissors and the underlying tissue was bluntly dissected until thesternohyoid and sternomastoid muscles were exposed. The left commoncarotid artery was located just below the region where the sternohyoidand sternomastoid meet. The vagus nerve was carefully separated from thecarotid artery. The left carotid artery was permanently occluded with ahigh-temp elecrocautery pen. Animals in which the common carotid arterywas not completely sealed or exhibited blood loss were immediatelyeuthanized. After a recovery period of 2-3 hrs the mice were placed in aglass cylinder vented with 8% oxygen balanced nitrogen flowing at a rateof 6 L/min. The glass cylinder was placed in a water bath at 36.5° C. tomaintain body temperature. After 50 min exposure to the low oxygenenvironment (8%) the mice were removed from the chamber and returned totheir cage. Mice were allowed to survive for 2 weeks following HI topermit the brain infarct in the ipsilateral hemisphere to develop beforeharvesting brain tissue for histological analysis.

Rotarod

The rotarod is a behavioral test that assesses motor performance inrodents. The apparatus consists of a rotating cylinder that mice walkalong. The rotational speed increases at a constant acceleration makingit more difficult for mice to continue walking. The amount of time spenton the rod (latency period for the animal to fall off the rod) wasrecorded as a measure of performance, with longer times indicative ofbetter motor performance. The acceleration of the rotarod was set to 100rot/min². Mice were tested on the third day of F5 treatment (24 hrspre-HI) and 2 weeks following HI (14 days post-HI). On each of thesedays the mice were tested with 3 sessions and the average time spent onthe rotarod was calculated for that day. The difference in performance14 days post-HI and 24 hrs pre-HI was determined and compared betweenthe two treatment groups.

Preparation of Tissue for Histology

Two weeks following HI the mice were humanely killed by an IP injectionof sodium pentobarbital at a dose of 240 mg/kg. The mice weretranscardially perfused with 0.9% saline then 4% paraformaldehyde (PFA)in phosphate buffer at a pH of 7.4. Post-fixation was achieved bystoring the brains for 48-72 hrs in 4% PFA. The tissue was cryoprotectedby submerging in a solution of 30% sucrose in 0.1 M phosphate buffer for24 hrs. Free floating coronal sections were cut on a freezing microtomeat a thickness of 30 μm and placed in a solution of phosphate bufferedsaline (PBS) with 5% sodium azide for long-term storage.

Nissl Staining

Sections 360 μm apart were mounted onto superforst glass slides anddried overnight. Serial brain sections about 30 μm thick were cut fromthe anterior and mid portion of the dorsal hippocampus. The sectionswere dehydrated using a graded ethanol series of increasing strength (2min of 50%, 70%, 95%, 100%) then incubated in xylenes for 5 min followedby rehydration using another graded series of ethanol of increasingdilution (100%, 95%, 70%, 50%). Brain sections were then rinsed in waterand incubated in a 1% cresyl violet solution for 10-15 min. The sectionswere rinsed and destained in a 1% acetic acid before being dehydratedthrough a graded series of ethanol solutions of increasing strength(50%, 70%, 95%, 100%). The tissue was cleared in xylenes thencoverslipped using Cytoseal. Images of the sections were captured usingPixeLink software with a 1× lens and a 10× objective. The images wereanalyzed using ImageJ software. The area of the hemisphere ipsilateralto common carotid artery occlusion and the area of the contralateralhemisphere were measured for all sections and a ratio ofipsilateral/contralateral area was calculated to determine hemisphereloss. A ratio of 1.0 indicated no hemisphere loss, while ratios lessthan 1.0 indicated loss.

Neuronal Nuclei (NeuN) Immunohistochemistry

To prepare tissue for immunohistochemical staining the brain sectionswere rinsed three times with PBS containing 0.1% Triton X (PBS-TX) forten minutes at room temperature (same for all subsequent washes). Thesections were then placed in PBS-TX containing 1% hydrogen peroxide for30 minutes to quench endogenous peroxidases. The tissue was again washedbefore being incubated in 5% horse serum in PBS-TX. Following incubationin serum the tissue was incubated with primary antibody at roomtemperature for one hour then shaken at 4° C. overnight. The primaryantibody was a monoclonal anti-NeuN antibody raised in mouse used at a1:2000 dilution in PBS-TX. After incubation overnight in primaryantibody, the tissue was washed and incubated for one hour in thesecondary antibody, anti-mouse raised in horse, used at a dilution of1:500. Another series of washes was performed and the tissue wasincubated in an Avidin-Biotin complex in PBS-TX at a dilution of 1:1000for one hour to amplify the signal of the secondary antibody. Thesections were washed and then placed in a solution of 0.5 mg/mldiaminobenzidine (DAB) with nickel, glucose oxidase, D-glucose, andammonium chloride in PBS. The tissue was reacted with the DAB solutionfor 5-10 minutes until staining of desired density was achieved. Thetissue was then washed in PBS, mounted on superfrost glass slides andleft overnight to dry. The sections were then dehydrated in a gradedethanol series (50%, 70%, 95%, and 100%), cleared in Xylene, andcoverslipped using Cytoseal. Sections stained for NeuN immunoreactivityin the striatum at 0.1 mm anterior to bregma and the hippocampus at 1.8mm posterior to bregma were captured on a light microscope usingPixeLink software at 50× (10× objective and a 5× lens). The images werethen analyzed using ImageJ software by an observer blind to thetreatment group. Cell counts in the striatum were obtained by firstconverting the image to an 8-bit grey scale. The binary tool wasselected so that only pixels above a threshold of 3× background wereblack on a white foreground. The striatum was outlined and thepositively labelled cells were counted using the analyze particlesfunction. An index of neuronal survival was calculated by dividing thenumber of NeuN positive cells in the ipsilateral striatum by the numberof NeuN positive cells in the contralateral striatum to yield a cellsurvival ratio. A value of 1.0 indicated no injury in the ipsilateralstriatum while a value of less than 1.0 indicated neuronal loss. Theimages of the hippocampus were converted to 8-bit grey scale and thebinary tool was selected so that only positively labelled cells wereblack on a white foreground. The hippocampus was outlined and the areaof labelled cells was measured with the measurement function. In thecase of the dorsal hippocampus, the dense packing of pyramidal neuronsprecluded cell counts in sections 30 μm thick. Neuronal loss wastherefore estimated by measuring the area occupied by NeuN positivecells in the entire hippocampus of sections cut at 1.8 mm posterior tobregma. The area occupied by NeuN positive neurons in the ipsilateralhippocampus was divided by the area occupied by NeuN positive neurons inthe contralateral hippocampus to obtain an index of neuronal loss forthis structure. An index of 1.0 indicated no neuronal loss in theipsilateral hippocampus, while values less than 1.0 indicated neuronalloss.

Results

In reference to FIG. 1, it can be seen that mice treated with F5 hadbetter motor performance as assessed by the rotarod compared to micetreated with vehicle.

Motor performance was assessed in the mice before and after HI as ameasure of neurological capacity. The difference in rotarod performance(14 days post-HI-24 hrs pre-HI) was greater for the group that wastreated with F5 than the control mice that received water. On average,mice in the F5 group improved their score following HI with amean±standard error of the mean (SEM) difference of 3.7±2.5 seconds (s).Mice treated with vehicle performed worse following HI with a mean±SEMdifference of −5.7±2.7 s. A two-sided student's t-test determined asignificant difference between these 2 group (t (45)=2.53; P=0.015). F5treatment therefore improved the neurological outcome following HIcompared to vehicle treatment.

In reference to FIG. 2, it can be seen that mice treated with F5 (50mg/kg/day for 3 days) were protected from the hemisphere loss caused byHI compared to vehicle treated mice.

As a result of the injurious effects of HI the brain hemisphere on theside ipsilateral to internal carotid cauterization becomes damaged andmay experience tissue loss. The mice treated with F5 experienced smallerhemisphere loss following HI with a mean±SEM ipsilateral/contralateralratio of 0.90±0.03 while mice treated with vehicle had a mean±SEM ratioof 0.80±0.03. The difference between groups was found to bestatistically significant using a two-sided student's t-test(t(45)=2.12; P=0.040). The apple fraction F5 was therefore protectiveagainst the infarction caused by HI.

In reference to FIG. 3, it can be seen that F5 treatment increased thenumber of viable neurons in the striatum after HI compared to vehicletreatment.

Cell death following HI occurs in several brain regions following HI.One region that is sensitive to neuronal death following HI is thestriatum. To assess neuronal loss following HI, brain sections werestained with the immunohistochemical marker NeuN to demark viableneurons. The number of neurons was counted in both the ipsilateral andcontralateral striata at 1.0 mm anterior to bregma. Theipsilateral/contralateral ratio was calculated to assess neuronal loss.It was found that mice given the F5 extract experienced a mean count±SEMof 0.73±0.08, while the mice treated with vehicle had a mean of0.47±0.08. The difference between the two groups was found to bestatistically significant using the two-sided student's t-test(t(45)=2.126; P=0.039). Therefore the apple extract was found toincrease the number of viable neurons in the striatum following HIcompared to the vehicle treated group.

Another brain region where neuronal death occurs is the hippocampus. Toassess the cell loss in the hippocampus following HI the brain sectionswere stained immunohistochemically with NeuN. The area of NeuN positiveneurons was measured in both the ipsilateral and contralateralhippocampus at 1.8 mm anterior to bregma. A ratio was determined bydividing the area of NeuN positive staining in the ipsilateralhippocampus divided by the area of staining in the contralateralhemisphere. In reference to FIG. 4, it can be seen that F5 treatment didnot significant prevent cell loss in the hippocampus in comparison tothe vehicle control group. The mice treated with F5 had a mean±SEM ratioof 0.63±0.09, while the vehicle control group showed a mean±SEM of0.45±0.09. The difference between the two groups was not found to besignificant, with a P value of 0.1771 (determined by a two sidedstudent's t-test). The results suggest that F5 treatment was notprotective in the hippocampus under the parameters tested. Furtherstudies will explore whether different dosages or treatment regimes mayextend protection to the hippocampus region.

The results to date suggest that 3 doses of F5 prior to HI isneuroprotective against tissue loss, as well as in protecting againstthe motor performance deficits experienced by those mice in the vehicletreatment group following HI.

Representative Photos are shown in FIGS. 5 and 6. Images selected werethe animal with the median score for respective group.

Example 3 F4 Reduces Death of Primary Cortical Neurons Subjected toOxygen Glucose Deprivation when Compared to Two F4 Components and ThreeF4 Metabolites

Methods

Lactate dehydrogenase is a stable cytosolic enzyme that is released bynecrotic cells upon membrane damage. The membrane integrity of corticalneurons was assayed by measuring the release of lactate dehydrogenase(LDH) using the Cytotoxicity Detection Kit^(PLUS) (Roche AppliedScience). This assay kit detects LDH released into culture supernates bya coupled enzymatic reaction. Positive (100% LDH release) and negative(spontaneous LDH release) controls were prepared in triplicate accordingto the manufacturer's instructions. Primary cortical neuron cultureswere prepared from cerebral cortices of embryonic day 16 CD1 mouseembryos. Cortical neuron cultures were exposed to vehicle (0.1% dimethylsulfoxide (DMSO)) or F4 at concentrations of 1, 0.1 or 0.01 μg/mL inserum-free conditions for a period of 12 h before they were subjected tooxygen glucose deprivation (OGD). The neurons were exposed to vehicle orF4 at concentrations of 1, 0.1 or 0.01 μg/mL phenolics throughout the 12h period of OGD. Following, cell culture supernates were collected fordetermination of released LDH. Absorbance was measured at 490 nm with areference wavelength of 620 nm. Percentage of total LDH release wascalculated by following the instructions provided by the manufacturer.Background was subtracted and LDH release in each sample was expressedas a percentage of the positive control.

In reference to FIG. 7, it can be seen that F4 significantly reduces thedeath of primary cortical neurons subjected to oxygen glucosedeprivation whereas no significant reduction was seen when cells weretreated with two active components of F4 (Q and Q3G) or threemetabolites (Q3′ S, Q3G1u, IR3G1cA). Primary cortical cultures that wereexposed to 1 μg/mL of F4 were significantly protected from necrotic celldeath induced by OGD (p<0.0001), as compared with vehicle-treated cells.F4 had a direct neuroprotective effect on cortical neurons underoxidative stress.

The results demonstrate that F4 is able to reduce neuronal cell loss inan experimental model of stroke (hypoxic-ischemic brain injury) at leastin part by directly protecting neurons from the damaging effects ofischemia (no glucose, no oxygen).

Example 4 F4 is Protective Against Hypoxic-Ischemic (HI) Brain Damage InVivo

The Methods for hypoxic-ischemic (HI) brain injury; NeuN staining, animage analysis are the same described in the examples above.

This study was conducted to explore optimal dosing parameters of F4 inreducing the damaging effects of HI brain injury in two vulnerableforebrain structures—dorsal hippocampus and striatum. For bothstructures, an oral (p.o.) dose of F4 at 25 mg/kg, given once daily for3 days prior to HI (FIGS. 8-11), produced significant and dramaticneuroprotection. Lower doses (5-10 mg/kg, p.o.) of F4 were lessneuroprotective than 25 mg/kg (p.o.) while 50 mg/kg (p.o.) of F4 was nomore effective than 25 mg/kg (p.o.). In terms of dose frequency, asingle oral administration of F4 (25 mg/kg) 24 hours before HI did notattenuate HI-induced injury in the striatum (FIG. 6). Oraladministration of F4 (25 mg/kg/day) for 3 days produced a reduction ofneuron loss in the striatum (FIG. 7) that was not further enhanced byincreasing the number of doses to 7 (FIG. 8). In the case of thehippocampus, a single dose of F4 (25 mg/kg, p.o.) did not reduce tissuedamage (FIG. 9). Three doses of F4 (25 mg/kg, p.o.) produced a reductionof HI-induced hippocampal atrophy but some neuronal loss was stillapparent with this dosing regime (FIG. 10). Seven doses of F4 (25 mg/kg,p.o.) did, however, prevent the loss of hippocampal neurons in thisstructure (FIG. 11). These differences likely reflect the fact that thehippocampus is more vulnerable to HI damage than the striatum. Relativeto the striatum, longer treatment with F4 (25 mg/kg/day for 7 daysrather than 3 days) resulted in maximal protection of the hippocampus.These findings indicate that for F4-induced neuroprotection, 25 mg/kg(p.o.) given once a day for 7 days was the optimal dosing regime in thismouse model of HI-induced brain injury.

In reference to FIG. 8, it can be seen that there is a dose-dependentreduction in HI-induced hippocampal neuron loss produced by oral (p.o.)administration of F4. NeuN staining is shown in the hippocampusipsilateral (A, C, E, G, I) and contralateral (B, D, F, H, J) of animalsexposed to 50 min of hypoxia-ischemia (HI) that received water orincreasing doses of the apple peel fraction F4. Five groups, composed of7-9 adult male C57B1/6 mice each, were dosed orally (p.o.) once a dayfor 3 days with water (10 ml/kg) or F4 (5, 10, 25 or 50 mg/kg). Allanimals received HI 24 hours after the last administration of water orF4. F4 produced a dose-dependent reduction of tissue atrophy andneuronal loss (NeuN immunoreactive cells) in the ipsilateralhippocampus. Administration of F4 at a dose of 5 mg/kg (p.o.) did notreduce the damaging effects of HI in the ipsilateral (C) hippocampus.Administration of the 10 mg/kg (p.o.) dose of F5 partially protected theipsilateral hippocampus (E) against brain injury caused by HI.Administration of 25 mg/kg (p.o.) (G) or 50 mg/kg (p.o.) (I) of F4appeared to produce a near complete protection against HI-inducedhippocampal injury HI.

In reference to FIG. 9, a dose-dependent reduction of HI-inducedhippocampal damage by F4 can be seen. Water (10 ml/kg) or F4 (5, 10, 25or 50 mg/kg) was administered once a day for 3 days prior to 50 minutesof HI. The last dose occurred 24 hours before HI. F4 produced a dosedependent reduction in hippocampal tissue loss. One-way ANOVA followedby Newman-Keuls comparison test. *p<0.05 relative to vehicle and F4 (5mg/kg, p.o.) groups.

In reference to FIG. 10, a dose-dependent reduction of HI-inducedstriatal neuron loss produced by F4 can be seen. NeuN staining is shownin the striatum ipsilateral (A, C, E, G, I) and contralateral (B, D, F,H, J) of animals exposed to 50 min of hypoxia-ischemia (HI) thatreceived water or increasing doses of the apple peel fraction F4. Fivegroups, composed of 7-9 adult male C57B1/6 mice each, were dosed orally(p.o.) once a day with water (10 ml/kg) or F4 (5, 10, 25 or 50 mg/kg).All animals received HI 24 hours after the last administration of wateror F4. F4 produced a dose-dependent reduction of tissue atrophy andneuronal loss (NeuN immunoreactive cells) in the ipsilateral striatum.Administration of F4 at a dose of 5 mg/kg (p.o.) did not reduce thedamaging effects of HI in the ipsilateral (C) striatum. Administrationof the 10 mg/kg (p.o.) dose of F4 partially protected the ipsilateralstriatum (E) against brain injury caused by HI. Administration of 25mg/kg (p.o.) (G) or 50 mg/kg (p.o.) (I) of F4 appeared to produce a nearcomplete protection against HI-induced striatal injury HI.

In reference to FIG. 11, a dose-dependent reduction of HI-inducedstriatal neuron loss is produced by F4. Water (10 ml/kg) or F4 (5, 10,25 or 50 mg/kg) was administered once a day for 3 days prior to 50minutes of HI. The last dose occurred 24 hours before HI. F4 produced adose dependent reduction of HI-induced neuron loss in the ipsilateralstriatum. One-way ANOVA followed by Newman-Keuls comparison test.*p<0.05 relative to vehicle and F4 (5 mg/kg, p.o.) groups.

In reference to FIG. 12. Failure of one dose of F4 (25 mg/kg, p.o.)before HI to reduce striatal neuron loss. Two groups of mice receivedwater (10 ml/kg, p.o.) or 25 mg/kg (p.o.) of F4 and were subjected to 50min of HI 24 hours later. Quantification of the relative number of NeuNpositive neurons in the ipsilateral and contralateral striatum revealedthat one dose of F4 (25 mg/kg, p.o.) failed to reduce striatal injury.Representative brain sections show NeuN immunoreactivity in theipsilateral (A,C) and contralateral (B,D) striatum of animals thatreceived water (A, B) or F4 (C,D) 14 days after HI.

In reference to FIG. 13, it can be seen that there is a reduction ofstriatal neuron loss by three doses of F4 (25 mg/kg/day, p.o) before HI.Two groups of mice received water (10 ml/kg, p.o.) or 25 mg/kg (p.o.) ofF5 once a day for 3 days. All animals were subjected to 50 min of HI 24hours after the last administration of water or F4. Quantification ofthe relative number of NeuN positive neurons in the ipsilateral andcontralateral striatum revealed that 3 doses of F4 (25 mg/kg, p.o.)reduced striatal injury. Representative brain sections show NeuNimmunoreactivity in the ipsilateral (A,C) and contralateral (B,D)striatum of animals that received water (A, B) or F4 (C,D) 14 days afterHI. *P<0.05, Mann-Whitney test.

In reference to FIG. 14, there was a reduction of striatal neuron lossby seven doses of F4 (25 mg/kg/day, p.o) before HI. Two groups of micereceived water (10 ml/kg, p.o.) or 25 mg/kg (p.o.) of F4 once a day for7 days. All animals were subjected to 50 min of HI 24 hours after thelast administration of water or F4. Quantification of the relativenumber of NeuN positive neurons in the ipsilateral and contralateralstriatum revealed that 7 doses of F4 (25 mg/kg, p.o.) reduced striatalinjury. Representative brain sections show NeuN immunoreactivity in theipsilateral (A,C) and contralateral (B,D) striatum of animals thatreceived water (A, B) or F5 (C,D) 14 days after HI. *P<0.05,Mann-Whitney test.

In reference to FIG. 15, it can be seen that one dose of F4 (25 mg/kg,p.o.) before HI did not significantly reduce hippocampal damage. Twogroups of mice received water (10 ml/kg, p.o.) or 25 mg/kg (p.o.) of F5and were subjected to 50 min of HI 24 hours later. The ability of F4 topreserve hippocampal tissue was estimated by taking a ratio of the areaof this structure in the ipsilateral and contralateral hemispheres ofsections stained for NeuN immunoreactivity. These measurements revealedthat a single dose of F4 (25 mg/kg, p.o.) failed to reduce hippocampalinjury. Representative brain sections show NeuN immunoreactivity in theipsilateral (A,C) and contralateral (B,D) hippocampus of animals thatreceived water (A, B) or F5 (C,D) 14 days after HI.

In reference to FIG. 16, it can be seen that three doses of F4 (25mg/kg, p.o.) before HI reduced hippocampal tissue loss. Two groups ofmice received water (10 ml/kg, p.o.) or 25 mg/kg (p.o.) of F4 once a dayfor 3 days. All animals were subjected to 50 min of HI 24 hours afterthe last administration of water or F4. The ability of F4 to preservehippocampal tissue was estimated by taking a ratio of the area of thisstructure in the ipsilateral and contralateral hemispheres of sectionsstained for NeuN immunoreactivity. These measurements revealed that 3doses of F4 (25 mg/kg, p.o.) reduced hippocampal injury. Representativebrain sections show NeuN immunoreactivity in the ipsilateral (A,C) andcontralateral (B,D) hippocampus of animals that received water (A, B) orF5 (C,D) 14 days after HI. *P<0.05, Mann-Whitney test.

In reference to FIG. 17, it can be seen that seven doses of F4 (25mg/kg, p.o.) before HI reduced hippocampal tissue loss. Two groups ofmice received water (10 ml/kg, p.o.) or 25 mg/kg (p.o.) of F4 once a dayfor 7 days. All animals were subjected to 50 min of HI 24 hours afterthe last administration of water or F4. The ability of F4 to preservehippocampal tissue was estimated by taking a ratio of the area of thisstructure in the ipsilateral and contralateral hemispheres of sectionsstained for NeuN immunoreactivity. These measurements revealed that 7doses of F4 (25 mg/kg, p.o.) reduced hippocampal injury. Representativebrain sections show NeuN immunoreactivity in the ipsilateral (A,C) andcontralateral (B,D) hippocampus of animals that received water (A, B) orF5 (C,D) 14 days after HI. *P<0.05, Mann-Whitney test.

With regard to the studies that showed no significant protection with asingle dose of F4 at 25 mg/kg, it is possible that a higher dose or adifferent route of administration may have resulted in a significantprotective effect. The results do nonetheless show significantprotection of treatment with F4 or F5 when administered at a suitabledose or dosing frequency, which can be determined by those of skill inthe art.

Example 5 F4 is Effective in an Animal Model of Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a widely acceptedanimal model for multiple sclerosis (MS). Immunization of mice with aportion of myelin oligodendrocyte glycoprotein (MOG) encompassing aminoacids 35-55 (MOG₃₅₋₅₅) in conjunction with Complete Freund's adjuvantresults in the development of paralysis associated with neuropathologyresembling MS. The present study performed to determine if oraladministration of F4 after the first clinical signs of EAE were apparent(hooked tail) reduced disease progression compared to animals thatreceived an equivalent amount of water. Disease progression wasdetermined by assessment of the clinical signs of EAE (paralysis,walking deficits) as described below.

Methods

Female adult C57B1/6 mice (Charles River; St. Constant, QU) 6-8 weeksold were immunized with MOG₃₅₋₅₅ (Sheldon Biotechnology Centre,Montreal, PQ) dissolved in 1×PBS (pH=7.4) emulsified in a 1:1 ratio withComplete Freund's adjuvant (CFA) containing 0.5 mg of Mycobacteriumtuberculosis H37RA (Difco Laboratories; BD Diagnostics). On day 0, theMOG₃₅₋₅₅ emulsion was injected subcutaneously (s.c.) bilaterally at thebase of the tail (300 μg/mouse). Pertussis toxin (PTX) (Sigma, St.Louis, Mo.), an immune booster, was diluted in saline and administeredintraperitoneally (i.p.) (300 ng/mouse) on day 0, and again on day 2.

A health check was carried out on day 5 and mice were randomly assignedto either a treatment group (n=10) or a water group (n=10). The weightsand clinical scores of each individual mouse were recorded daily over 31days beginning on day 7. Drug (F4) or water was administered orally (25mg/kg) immediately upon initial presentation of clinical signs anddosing continued daily for the remainder of the experiment.

The following grading scheme was used to clinically score the animals:0, no clinical signs; 0.5, hooked tail; 1, hooked tail with splay; 1.5,flaccid tail with splay; 2, beginning of walking deficits/minor ataxia;2.5, severe walking deficits; 3, dropped pelvis in addition to severewalking deficits; 3.5, unilateral hindlimb paralysis; 4, bilateralhindlimb paralysis; 5, moribund. Mice were supplied with mash andhandfed Neutri-cal® (Evsco Pharmaceuticals; Buena, N.J.) when they wereno longer able to reach food. Lactated ringers solution (50 U/Daily) wasprovided when a mouse reached a score≧2.5 or when their weight fell 10%below baseline. All clinical scores were recorded by a blinded scorer.

Results

Treatment with F4 (n=9) led to a significant reduction in clinicalimpairment compared to treatment with water alone (n=10; 2 way ANOVAfollowed by Bonferroni tests, p<0.01). One mouse was removed from thestudy (F4 group) due to health concerns that were unrelated to theexperiment. From days 19-22, the average clinical score in the F4-EAEgroup peaked at ˜2.5, while maximal increases in disease severity forthe water-EAE group ranged from ˜3.5-4. Both groups remitted; however,the F4 group recovered more rapidly and showed a trend toward completerecovery while the water group began to relapse on day 31.

The ability of F4 to reduce the severity of EAE in mice suggests that ithas the potential to halt disease progression in patients with multiplesclerosis, an important facet of MS treatment.

In reference to FIG. 18, it can be seen that F4 significantlyameliorates clinical severity in EAE. Female C57B1/6 mice were immunizedwith MOG₃₅₋₅₅ (300 μg/mouse) in a 1:1 ratio with CFA on day 0 andboosted with pertussis toxin injections (300 ng/mouse) on days 0 and 2.The weights and clinical scores of each individual mouse were recordeddaily over 31 days beginning on day 7. Oral administration of F4 (25mg/kg) or vehicle (water 10 ml/kg) was administered 24 hours after thefirst presentation of clinical signs and dosing continued daily for theremainder of the experiment. By day 10, all mice presented with clinicalsigns and were being dosed with their respective treatment (downwardarrow). The following grading scheme was used to clinically score theanimals: 0, no clinical signs; 0.5, hooked tail; 1, hooked tail withsplay; 1.5, flaccid tail with splay; 2, beginning of walking deficitsand ataxia; 2.5, severe walking deficits; 3, dropped pelvis in additionto severe walking deficits; 3.5, unilateral hindlimb paralysis; 4,bilateral hindlimb paralysis; 5, moribund. Relative to EAE-water group(n=10), EAE-F4 animals (n=9) displayed less severe clinical scores anddid not relapse (2 way ANOVA followed by Bonferroni tests, p<0.01). Fromdays 19-22 the average clinical score in the F4-EAE group peaked at˜2.5, while maximal increases in disease severity for the water-EAEgroup ranged from ˜3.5-4.

Example 6 F4 Reduces LPS-Induced TNF-α Release

Methods

Two groups, composed of 15 adult male C57BL/6 (25 g) mice each, weredosed orally with water (0.01 ml/g) or F4 (50 mg/kg) every 24 h for 3days. Twenty-four hours after the last administration of water of F4,animals from each of these two groups were subject to either sham HIsurgery (n=10) or 50 min of HI (n=5). Either 16 hours later (no injury)or 6 hours later (injury), whole blood was collected into microtainerscontaining lithium heparin (BD Biosciences, Franklin Lakes, N.J.) fromall mice. LPS (lipopolysaccharide, serotype 0111:B4; Sigma-Aldrich, St.Louis, Mo.) derived from Escherichia coli (100 μg) was used to inducethe release of TNF-α, a surrogate marker for activation of thepro-inflammatory enzyme phosphodiesterase 4 mouse whole blood accordingto Moore et al. (2006). Freshly prepared LPS was prepared at aconcentration of 5 mg/ml in 0.1% bovine serum albumin inphosphate-buffered saline and diluted to a final concentration of 100 μgof LPS/ml of blood. The blood was incubated for 4 h at 37° C. in ahumidified tissue culture incubator supplemented with 5% CO₂. Followingthis incubation, blood was centrifuged at 1400 g for 10 min at 4° C.,and the plasma was collected and stored at −80° C. LPS-induced TNF-αrelease in whole blood was assessed using an ELISA and performedaccording to the manufacturer's protocol (BioSource International,Camarillo, Calif.). In brief, using a solid-phase sandwich ELISA and astreptavidin-peroxidase reaction, the intensity of the colored productis directly proportional to the amount of TNF-α in the sample. Theabsorbance was read at 450 nm blanked against a chromagen blank, and theamount of TNF-α in each sample was calculated using a recombinant mouseTNF-α.

Results

In reference to FIG. 19, A, LPS-induced TNF-α release was the same inwhole blood from sham treated animals (no brain injury) who receivedoral administration of water or F4. B, LPS-induced TNF-α release was thegreater in whole blood from HI animals (brain injury) that received oraladministration of water compared to mice that received F4. These resultssuggests that F4 has a potent anti-inflammatory effect in the presenceof brain injury but, advantageously, will not act as a generalimmunosuppressant in healthy individuals.

CONCLUSIONS

These results suggest that F4 reduces HI brain injury by decreasingproduction of the pro-inflammatory cytokine TNF-α in whole blood thatwould otherwise mobilize the innate immune system resulting ininfiltration of damaging immune cells such as macrophages andneutrophils in to the central nervous system. Flavonoids such asquercetin are known to block the phosphodiesterase 4 (Pleuso, 2006).Since LPS-induced TNF-α is inhibited in whole blood from animals treatedwith doses of selective phosphodiesterase 4 inhibitors block doses thatprevent the clinical signs of EAE (Moore et al, 2006), our resultsindicate the F4 reduced neurodegeneration in the HI and EAE models byinhibiting this pro-inflammatory enzyme.

It should be noted that the results for Fractions F4 and F5 were similarin all studies where both fractions were tested.

The above-described embodiments are intended to be examples only.Alterations, modifications and variations can be effected to theparticular embodiments by those of skill in the art without departingfrom the scope of the disclosure, which is defined solely by the claimsappended hereto.

REFERENCES

-   Heo H J, Kim D O, Choi S J, Shin D H and Lee C Y, 2004. Apple    phenolics protect in vitro oxidative stress-induced neuronal cell    death. J Food Sci 69, S357-S360-   Gugler R et al. 1975 Eur. J. Clin. Pharmacol. 9: 229-   Huber G. M. and Rupasinghe, 2009. Phenolic Profiles and Antioxidant    Properties of Apple Skin Extracts. J Food Chem, 74(9): C693-C700.-   Kaur and Kapoor, 2001. Antioxidants in fruits and    vegetables—millenium's health. International J Food Sci Tec 36,    703-725.-   Kim et al., 2005. Phenolic Extraction from Apple Peel by Cellulases    from Thermobifidia fusca. J Agric Food Chem, 53, 9560-9565.-   Kondo et al., 2002. Antioxidative activity of apple skin or flesh    extracts associated with fruit development on selected apple    cultivars. Sci Horticult 96(1-4), 177.-   Levine, S., 1960. Anoxic-ischemic encephalopathy in rats. Am J.    Pathol. 36, 1-17.-   C. S. Moore, N. Earl, R. Frenette, A. Styhler, J. A. Mancini, D. W.    Nicholson, A. L. O. Hebb, T. Owens, and G. S. Robertson 2006 Journal    of Pharmacology and Experimental Therapeutics 319, 63-72-   Pleuso M.R. Exp Biol Med 231:1287-1299, 2006-   Rupasinghe, H.P.V., N. Erkan, and A. Yasmin. 2010. Antioxidant    protection of eicosapentaenoic acid and fish oil oxidation by    polyphenolic-enriched apple skin extract. J. Agric. Food Chem.    58:1233-1239.-   Rutz, M. J. et al. 2009. Dietary administration of high doses of    pterostilbene and quercetin to mice is not toxic. J. Agric. Food    Chem 57(8): 3180-6.-   Soundararajan et al. 2008. Quercetin 3-glucoside protects    neuroblastoma (SYH-SY5Y) cells in vitro against oxidative damage by    inducing sterol regulatory element-binding protein-2-mediated    cholesterol biosynthesis. JBC, January 25, 283 (34), 2231-2245.-   Williams R J et al. Free Radical Biology and Medicine. Volume 36,    Issue 7, 1 Apr. 2004, Pages 838-849-   Wolfe K L, Liu R H 2003. Apple peels as a value-added food    ingredient. J Argic Food Chem, 51(6), 1676-83.-   Wolfe K, Wu X, Liu R H 2003. Antioxidant activity of apple peels. J    Agric Food Chem, 51, 609-614.

All referenced documents are incorporated herein in their entirety

1. A composition for use in preventing or treating a disease orcondition associated with oxidative stress or inflammation, thecomposition comprising a phenolic extract or fraction thereof derivedfrom apple skin.
 2. The composition of claim 1, wherein the phenolicextract or fraction thereof comprises a flavonol component, ananthocyanin component, a dihydrochalcone component, a phenolic acidcomponent, and a flavan-3-ol component.
 3. The composition of claim 2,wherein the flavonol component comprises quercetin, Q-3-O-paltoside,Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnosideor a combination thereof.
 4. The composition of any one of claims 2 to3, wherein the anthocyanin component comprises cyanidin-3-O-galactoside.5. The composition of any one of claims 2 to 4, wherein thedihydrochalcone component comprises phloridzin, phloritin or acombination thereof.
 6. The composition of any one of claims 2 to 5,wherein the phenolic acid component comprises chlorogenic acid, cafeicacid, ferulic acid, isoferulic acid or a combination thereof.
 7. Thecomposition of any one of claims 2 to 6, wherein the flavan-3-olcomponent comprises epigallocatechin, catechin, epicatechin or acombination thereof.
 8. The composition of any one of claims 1 to 7,wherein the extract or fraction thereof comprises between about 60.0% toabout 95.0% flavonol.
 9. The composition of any one of claims 1 to 8,wherein the extract or fraction thereof comprises between about 1.0% toabout 20.0% flavan-3-ol.
 10. The composition of any one of claims 1 to9, wherein the extract or fraction thereof comprises between about 1.0%to about 20.0% phenolic acid.
 11. The composition of any one of claims 1to 10, wherein the extract or fraction thereof comprises between about0.5% to about 10.0% dihydrochalcone.
 12. The composition of any one ofclaims 1 to 11, wherein the extract or fraction thereof comprisesbetween about 0.5% to about 10.0% cyanidin-O-galactoside.
 13. Thecomposition of any one of claims 1 to 12, wherein the phenolic extractor fraction thereof comprises: a flavonol component comprisingquercetin, Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside,Q-3-O-glucoside and Q-3-O-rhamnoside; an anthocyanin componentcomprising cyanidin-3-β-galactoside; a dihydrochalcone componentcomprising phloridzin and phloritin; a phenolic acid componentchlorogenic acid, cafeic acid, ferulic acid and isoferulic acid; and aflavan-3-ol component comprising epigallocatechin, catechin andepicatechin.
 14. The composition of claim 1, wherein the extract orfraction thereof comprises: from about 10.0% to about 60.0%, from about15.0% to about 50.0%, from about 20.0% to about 50.0%, from about 20.0%to about 35.0%, from about 20.0% to about 30.0%, from about 20.0% toabout 25.0%, Q-3-O-galactoside; from about 10.0% to about 60.0%, fromabout 15.0% to about 50.0%, from about 20.0% to about 50.0%, from about20.0% to about 40.0%, from about 20.0% to about 35.0%, from about 20.0%to about 30.0%, from about 20.0% to about 25.0%, from about 30.0% toabout 35.0%, Q-3-O-rhamnoside; from about 1.0% to about 20.0%, fromabout 5.0% to about 15.0%, from about 7.0% to about 13.0%, from about5.0% to about 10.0%, from about 10.0% to about 15.0%, Q-3-O-rutinoside;from about 1.0% to about 20.0%, from about 5.0% to about 15.0%, fromabout 7.0% to about 13.0%, from about 10.0% to about 15.0%, from about10.0% to about 15.0%, Q-3-O-glucoside; from about 0.5% to about 10.0%,from about 0.5% to about 5.0%, from about 0.5% to about 2.5, from about0.5% to about 2.0%, from about 1.0% to about 2.0%, from about 2.0% toabout 6.0%, from about 3.5% to about 5.5%, from about 4.0% to about5.0%, cyanidin-3-O-galactoside; from about 0.5% to about 10.0%, fromabout 1.0% to about 10.0%, from about 2.0% to about 10.0%, from about1.0% to about 5.0%, from about 1.5% to about 4.5%, from about 3.5% toabout 7.5%, about 3.0% to about 6.0%, about 3%, about 5% phloridzin;from about 1.0% to about 20.0%, from about 2.0% to about 15.0%, about5.0% to about 15.0%, from about 2.5% to about 6.5%, from about 8% toabout 12.0%, about 4% or about 10% chlorogenic acid; and from about 1.0%to about 20.0%, from about 1.0% to about 15.0%, from about 5.0% to about15.0%, from about 2.5% to about 10.0%, from about 2.5% to about 6.5%,from about 5.0% to about 10.0%, from about 8.0% to about 10.0%epicatechin.
 15. The composition of claim 14, which comprises: fromabout 20.0% to about 30.0% Q-3-β-galactoside; from about 20.0% to about30.0% Q-3-O-rhamnoside; from about 10.0% to about 15.0%,Q-3-O-rutinoside; from about 10.0% to about 15.0%, Q-3-O-glucoside; fromabout 2.0% to about 6.0% cyanidin-3-O-galactoside; from about 1.0% toabout 5.0%, phloridzin; from about 8% to about 12.0% chlorogenic acid;and from about 5.0% to about 10.0% epicatechin.
 16. The composition ofclaim 14, which comprises: from about 20.0% to about 35.0%Q-3-O-galactoside; from about 20.0% to about 35.0% Q-3-O-rhamnoside;from about 5.0% to about 15.0% Q-3-O-rutinoside; from about 5.0% toabout 15.0% Q-3-O-glucoside; from about 0.5% to about 2.5cyanidin-3-O-galactoside; from about 1.0% to about 5.0% phloridzin; fromabout 5.0% to about 15.0% chlorogenic acid; and from about 5.0% to about10.0% epicatechin.
 17. The composition of claim 1, wherein the phenolicextract or fraction thereof comprises: a flavonol component comprisingquercetin, Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside,Q-3-O-glucoside and Q-3-O-rhamnoside; an anthocyanin componentcomprising cyanidin-3-O-galactoside; a dihydrochalcone componentcomprising phloridzin and phloritin; a phenolic acid componentchlorogenic acid, cafeic acid, ferulic acid and isoferulic acid; and aflavan-3-ol component comprising epigallocatechin, catechin andepicatechin.
 18. The composition of any one of claims 1 to 13, whereinthe phenolic extract is obtainable by an aqueous extraction processhaving the following steps: obtaining a sample of apple skins; treatingthe skins to inhibit degradation of phenolic compounds; optionallydehydrating the skins and converting the skins to a powder form;extracting the skins one or more times with a food-grade solvent, suchas, ethanol; optionally subjecting the skins to sonication duringextraction; removing solids to obtain a phenolic extract; optionallyconcentrating the phenolic extract; optionally removing sugars from thephenolic extract, and optionally concentrating, drying and/or freezingthe phenolic extract.
 19. The composition of claim 18, wherein thephenolic extract is purified.
 20. The composition of claim 18 or 19,wherein the fraction is obtainable by fractionation of the phenolicextract using a suitable eluent, followed by selection of a fractionhaving a high phenolic content, in particular, a high flavonol content.21. The composition of claim 20, wherein the eluent is ethanol andwherein the chromatography is flash chromatography with a C18 columnusing a polymeric sorbent.
 22. The composition of claim 20 or 21,wherein the fraction is eluted in about 40% to about 60% ethanol. 23.The composition of claim 22, wherein the fraction is eluted in about 45%to about 50% ethanol.
 24. The composition of any one of claims 18 to 23,wherein the fraction has the following phenolic profile: from about60.0% to about 95.0% flavonol selected from the group consisting ofquercetin, Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside,Q-3-O-glucoside, Q-3-O-rhamnoside and combinations thereof; from about0.5% to about 10.0% cyanidin-3-O-galactoside; from about 1.0% to about10.0% dihydrochalcone selected from the group consisting of phloridzin,phloritin and combinations thereof; from about 1.0% to about 20.0%phenolic acid selected from the group consisting of chlorogenic acid,cafeic acid, ferulic acid, isoferulic acid and combinations thereof; andfrom about 1.0% to about 20.0% flavan-3-ol selected from the groupconsisting of epigallocatechin, catechin, epicatechin and combinationsthereof, wherein the percentages are based the total weight of phenoliccontent of the fraction and wherein the total does not exceed 100%. 25.The composition of claim 24, wherein the fraction has the followingphenolic profile: from about 70.0% to about 90.0% flavonol selected fromthe group consisting of quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside and combinationsthereof; from about 0.5% to about 5.0% cyanidin-3-O-galactoside; fromabout 2.0% to about 10.0% dihydrochalcone selected from the groupconsisting of phloridzin, phloritin and combinations thereof; from about2.0% to about 15.0% phenolic acid selected from the group consisting ofchlorogenic acid, cafeic acid, ferulic acid, isoferulic acid andcombinations thereof; and from about 1.0% to about 15.0% flavan-3-olselected from the group consisting of epigallocatechin, catechin,epicatechin and combinations thereof.
 26. The composition of claim 24,wherein the fraction comprises: from about 80.0% to about 90.0% flavonolselected from the group consisting of quercetin, Q-3-O-paltoside,Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnosideand combinations thereof; from about 0.5% to about 2.5%cyanidin-3-O-galactoside; from about 3.5% to about 7.5% dihydrochalconeselected from the group consisting of phloridzin, phloritin andcombinations thereof; from about 2.5% to about 6.5% phenolic acidselected from the group consisting of chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid and combinations thereof; and from about2.5% to about 6.5% flavan-3-ol selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof. 27.The composition of claim 24, wherein the fraction comprises: from about70.0% to about 80.0% flavonol selected from the group consisting ofquercetin, Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside,Q-3-O-glucoside, Q-3-O-rhamnoside and combinations thereof; from about2.0% to about 6.0% cyanidin-3-O-galactoside; from about 1.5% to about4.5% dihydrochalcone selected from the group consisting of phloridzin,phloritin and combinations thereof; from about 8.0% to about 12.0%phenolic acid selected from the group consisting of chlorogenic acid,cafeic acid, ferulic acid, isoferulic acid and combinations thereof; andfrom about 8.0% to about 12.0% flavan-3-ol selected from the groupconsisting of epigallocatechin, catechin, epicatechin and combinationsthereof.
 28. The composition of any of claims 1 to 27, furthercomprising a pharmaceutically acceptable excipient.
 29. The compositionof any one of claims 1 to 19, wherein the disease or conditionassociated with oxidative stress and/or inflammation is aging, anautoimmune disorder, a neurodegenerative disorder, a metabolic disorderor a vascular disorder.
 30. The composition of claim 29, wherein thevascular disorder is stroke.
 31. The composition of claim 29, whereinthe neurodegenerative disease is multiple sclerosis, vascular dementia,Parkinson's disease, or Alzheimer's disease.
 32. A method of preventingor treating a disease or condition associated with oxidative stressand/or inflammation, comprising administering to a subject an effectiveamount of a composition of any one of claims 1 to
 28. 33. A method ofpreventing or treating a disease or condition associated with oxidativestress and/or inflammation, comprising administering to a subject aneffective amount of a phenolic extract or fraction thereof derived fromapple skin.
 34. The method of claim 33, wherein the phenolic extract orfraction thereof comprises a flavonol component, an anthocyanincomponent, a dihydrochalcone component, a phenolic acid component, and aflavan-3-ol component.
 35. The method of claim 34, wherein the flavonolcomponent comprises quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside or a combinationthereof.
 36. The method of claim 34 or 35, wherein the anthocyanincomponent comprises cyanidin-3-O-galactoside.
 37. The method of any oneof claims 34 to 36, wherein the dihydrochalcone component comprisesphloridzin, phloritin or a combination thereof.
 38. The method of anyone of claims 34 to 37, wherein the phenolic acid component compriseschlorogenic acid, cafeic acid, ferulic acid, isoferulic acid or acombination thereof.
 39. The method of any one of claims 34 to 38,wherein the flavan-3-ol component comprises epigallocatechin, catechin,epicatechin or a combination thereof.
 40. The method of claim 39,wherein the phenolic extract or fraction thereof comprises: a flavonolcomponent comprising quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside and Q-3-O-rhamnoside; an anthocyanincomponent comprising cyanidin-3-O-galactoside; a dihydrochalconecomponent comprising phloridzin and phloritin; a phenolic acid componentchlorogenic acid, cafeic acid, ferulic acid and isoferulic acid; and aflavan-3-ol component comprising epigallocatechin, catechin andepicatechin.
 41. The method of any one of claims 34 to 40, wherein thephenolic extract is obtainable by an aqueous extraction process havingthe following steps: obtaining a sample of apple skins; treating theskins to inhibit degradation of phenolic compounds; optionallydehydrating the skins and converting the skins to a powder form;extracting the skins one or more times with a food-grade solvent, suchas, ethanol; optionally subjecting the skins to sonication duringextraction; removing solids to obtain a solid free phenolic extract;optionally concentrating the phenolic extract; optionally removingsugars from the phenolic extract, and optionally concentrating, dryingand/or freezing the phenolic extract.
 42. The method of claim 41,wherein the phenolic extract is purified.
 43. The method of any one ofclaim 40 or 41, wherein the fraction is obtainable by fractionation ofthe phenolic extract in a chromatography column using a suitable eluent,followed by selection of a fraction having a high phenolic content, inparticular, a high flavonol content.
 44. The method of claim 43, whereinthe eluent is ethanol and wherein the chromatography is flashchromatography with a C18 column using a polymeric sorbent.
 45. Themethod of claim 43 or 44, wherein the fraction is eluted in about 40% toabout 60% ethanol.
 46. The method of claim 45, wherein the fraction iseluted in about 45% to about 50% ethanol.
 47. The method of any one ofclaims 34 to 47, wherein the fraction has the following phenolicprofile: from about 60.0% to about 95.0% flavonol selected from thegroup consisting of quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside and combinationsthereof; from about 0.5% to about 10.0% cyanidin-3-O-galactoside; fromabout 1.0% to about 10.0% dihydrochalcone selected from the groupconsisting of phloridzin, phloritin and combinations thereof; from about1.0% to about 20.0% phenolic acid selected from the group consisting ofchlorogenic acid, cafeic acid, ferulic acid, isoferulic acid andcombinations thereof; and from about 1.0% to about 20.0% flavan-3-olselected from the group consisting of epigallocatechin, catechin,epicatechin and combinations thereof, wherein the percentages are basedthe total weight of phenolic content of the fraction and wherein thetotal does not exceed 100%.
 48. The method of claim 47, wherein thefraction has the following phenolic profile: from about 70.0% to about90.0% flavonol selected from the group consisting of quercetin,Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside,Q-3-O-rhamnoside and combinations thereof; from about 0.5% to about 5.0%cyanidin-3-O-galactoside; from about 2.0% to about 10.0% dihydrochalconeselected from the group consisting of phloridzin, phloritin andcombinations thereof; from about 2.0% to about 15.0% phenolic acidselected from the group consisting of chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid and combinations thereof; and from about1.0% to about 15.0% flavan-3-ol selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof. 49.The method of claim 47, wherein the fraction comprises: from about 80.0%to about 90.0% flavonol selected from the group consisting of quercetin,Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside,Q-3-O-rhamnoside and combinations thereof; from about 0.5% to about 2.5%cyanidin-3-O-galactoside; from about 3.5% to about 7.5% dihydrochalconeselected from the group consisting of phloridzin, phloritin andcombinations thereof; from about 2.5% to about 6.5% phenolic acidselected from the group consisting of chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid and combinations thereof; and from about2.5% to about 6.5% flavan-3-ol selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof. 50.The method of claim 47, wherein the fraction comprises: from about 70.0%to about 80.0% flavonol selected from the group consisting of quercetin,Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside,Q-3-O-rhamnoside and combinations thereof; from about 2.0% to about 6.0%cyanidin-3-O-galactoside; from about 1.5% to about 4.5% dihydrochalconeselected from the group consisting of phloridzin, phloritin andcombinations thereof; from about 8.0% to about 12.0% phenolic acidselected from the group consisting of chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid and combinations thereof; and from about8.0% to about 12.0% flavan-3-ol selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof. 51.The method of any one of claims 34 to 50, wherein the subject issusceptible to or is suffering from a disease or condition associatedwith.
 52. The method of any one of claims 34 to 51, wherein the diseaseor condition associated with oxidative stress and/or inflammation isaging, an autoimmune disorder, a neurodegenerative disorder, a metabolicdisorder or a vascular disorder.
 53. The method of claim 52, wherein thevascular disorder is stroke.
 54. The method of claim 52, wherein theneurodegenerative disease is multiple sclerosis, vascular dementia,Parkinson's disease, or Alzheimer's disease.
 55. The method of any oneof claims 34 to 54, wherein the phenolic extract or fraction thereof isadministered to a subject in multiple doses.
 56. The method of any oneof claims 34 to 55, wherein the phenolic extract or fraction thereof isadministered orally.
 57. The method of any one of claims 34 to 56,wherein the phenolic extract or fraction thereof is administered in theform of a concentrate, a liquid, a powder, an emulsion, a suspension, apaste, a gel, a film, a gum, a drop, a tablet, a capsule, amicrocapsule, a food additive.
 58. A fraction of a phenolic apple skinextract, the extract obtainable by an aqueous extraction process, thefraction obtainable by fractionating the extract in a chromatographycolumn using a suitable eluent and selecting a fraction having a highphenolic content, wherein the fraction comprises a flavonol component,an anthocyanin component, a dihydrochalcone component, a phenolic acidcomponent, and a flavan-3-ol component.
 59. The fraction of claim 58,wherein the flavonol component comprises quercetin, Q-3-O-paltoside,Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnosideor a combination thereof; the anthocyanin component comprisescyanidin-3-O-galactoside; the dihydrochalcone component comprisesphloridzin, phloritin or a combination thereof; the phenolic acidcomponent comprises chlorogenic acid, cafeic acid, ferulic acid,isoferulic acid or a combination thereof; and the flavan-3-ol componentcomprising epigallocatechin, catechin and epicatechin.
 60. The fractionof claim 58, which has the following phenolic profile: from about 60.0%to about 95.0% flavonol selected from the group consisting of quercetin,Q-3-O-paltoside, Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside,Q-3-O-rhamnoside and combinations thereof; from about 0.5% to about10.0% cyanidin-3-O-galactoside; from about 1.0% to about 10.0%dihydrochalcone selected from the group consisting of phloridzin,phloritin and combinations thereof; from about 1.0% to about 20.0%phenolic acid selected from the group consisting of chlorogenic acid,cafeic acid, ferulic acid, isoferulic acid and combinations thereof; andfrom about 1.0% to about 20.0% flavan-3-ol selected from the groupconsisting of epigallocatechin, catechin, epicatechin and combinationsthereof, wherein the percentages are based the total weight of phenoliccontent of the fraction and wherein the total does not exceed 100%. 61.The fraction of claim 58, wherein the fraction has the followingphenolic profile: from about 70.0% to about 90.0% flavonol selected fromthe group consisting of quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside and combinationsthereof; from about 0.5% to about 5.0% cyanidin-3-O-galactoside; fromabout 2.0% to about 10.0% dihydrochalcone selected from the groupconsisting of phloridzin, phloritin and combinations thereof; from about2.0% to about 15.0% phenolic acid selected from the group consisting ofchlorogenic acid, cafeic acid, ferulic acid, isoferulic acid andcombinations thereof; and from about 1.0% to about 15.0% flavan-3-olselected from the group consisting of epigallocatechin, catechin,epicatechin and combinations thereof.
 62. The fraction of claim 58comprising: from about 80.0% to about 90.0% flavonol selected from thegroup consisting of quercetin, Q-3-O-paltoside, Q-3-O-rutinoside,Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnoside and combinationsthereof; from about 0.5% to about 2.5% cyanidin-3-O-galactoside; fromabout 3.5% to about 7.5% dihydrochalcone selected from the groupconsisting of phloridzin, phloritin and combinations thereof; from about2.5% to about 6.5% phenolic acid selected from the group consisting ofchlorogenic acid, cafeic acid, ferulic acid, isoferulic acid andcombinations thereof; and from about 2.5% to about 6.5% flavan-3-olselected from the group consisting of epigallocatechin, catechin,epicatechin and combinations thereof.
 63. The fraction of claim 58,wherein the fraction comprises: from about 70.0% to about 80.0% flavonolselected from the group consisting of quercetin, Q-3-O-paltoside,Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-rhamnosideand combinations thereof; from about 2.0% to about 6.0%cyanidin-3-O-galactoside; from about 1.5% to about 4.5% dihydrochalconeselected from the group consisting of phloridzin, phloritin andcombinations thereof; from about 8.0% to about 12.0% phenolic acidselected from the group consisting of chlorogenic acid, cafeic acid,ferulic acid, isoferulic acid and combinations thereof; and from about8.0% to about 12.0% flavan-3-ol selected from the group consisting ofepigallocatechin, catechin, epicatechin and combinations thereof. 64.The fraction of any one of claims 58 to 64 which is obtainable byelution in about 40% to about 60% ethanol.
 65. The fraction of any oneof claims 58 to 64 which is obtainable by elution in about 45% to about50% ethanol.
 66. A dietary supplement or natural health product forpreventing or reducing damage due to oxidative stress comprising thecomposition of any one of claims 1 to 31 or the fraction of any one ofclaims 58 to
 65. 67. The dietary supplement or natural health product ofclaim 68, which is in the form of a concentrate, a liquid, a powder, anemulsion, a suspension, a film, a paste, a gel, a gum, a drop, a tablet,a capsule, a microcapsule or a food additive.
 68. A functional food orbeverage comprising the composition of any one of claims 1 to 31 or thefraction of any one of claims 58 to
 65. 69. A pharmaceutical compositioncomprising the composition of any one of claims 1 to 31 or the fractionof any one of claims 58 to 65 together with a pharmaceuticallyacceptable excipient.
 70. The pharmaceutical composition of claim 69,which is formulated for enteral administration, topical administration,parenteral administration, intrapulmonary administration or nasaladministration.
 71. The pharmaceutical composition of claim 70 whereinthe enteral administration is oral administration.
 72. Thepharmaceutical composition of any one of claims 69 to 71 for use in theprevention or treatment of a disease or condition associated withoxidative stress and/or inflammation.
 73. The pharmaceutical compositionof claim 72, wherein the disease or condition associated with oxidativestress and/or inflammation is aging, an autoimmune disorder, aneurodegenerative disorder, a metabolic disorder or a vascular disorder.74. The pharmaceutical composition of claim 73, wherein the vasculardisorder is stroke.
 75. The pharmaceutical composition of claim 73,wherein the neurodegenerative disease is multiple sclerosis, vasculardementia, Parkinson's disease, or Alzheimer's disease.
 76. Use of thecomposition of any one of claims 1 to 31 or the fraction of any one ofclaims 32-57 for the preparation of a medicament for the treatment orprevention of a disease or condition associated with oxidative stressand/or inflammation.
 77. Use of the composition of any one of claims 1to 31 or the fraction of any one of claims 32-57 for the treatment orprevention of a disease or condition associated with oxidative stress.78. A commercial package comprising the composition of any one of claims1 to 31 or the fraction of any one of claims 32-57 together withinstructions for use as a dietary supplement or natural health product.79. A commercial package comprising the dietary supplement or naturalhealth product of any one of claims 66 to 68 together with instructionsfor use in promoting health.
 80. A commercial package comprising thepharmaceutical composition of any one of claims 69 to 75 together withinstructions for use in the use in the treatment or prevention of adisease or condition associated with oxidative stress and/orinflammation.
 81. A food additive comprising the fraction of any one ofclaims 58 to
 65. 82. A cosmetic product comprising the fraction of anyone of claims 58 to
 65. 83. The invention as hereinbefore described.